CFTR regulators and methods of use thereof

ABSTRACT

Provided herein are compounds that activate CFTR and methods for treating constipation, dry eye disorders, and other diseases and disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase of Application PCT/US2016/068569,filed Dec. 23, 2016, which claims priority from Application 62/387,590filed on Dec. 24, 2015 in the United States. The entire contents ofwhich is incorporated herein by reference in its entirety and for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with the government support under Grant Nos.TR000004, EY023981, EY013574, EB000415, DK035124, DK072517 and DK101373,awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Constipation is a common clinical complaint in adults and children thatnegatively impacts quality of life. The prevalence of chronicconstipation has been estimated to be 15% in the U.S. population, withhealth-care costs estimated at approximately 7 billion dollars annually,with in excess of 500 million dollars spent on laxatives. The mainstayof constipation therapy includes laxatives and many of them areavailable over the counter (soluble fiber, polyethylene glycol,probiotics, etc.). There are two FDA-approved chloride channelactivators, lubiprostone and linaclotide, for treatment of constipation,but clinical trials showed variable and unimpressive efficacy of bothdrugs. Despite the wide range of therapeutic options, there is acontinued need for safe and effective drugs to treat constipation.

Dry eye is a heterogeneous tear film disorder that results in eyediscomfort, visual disturbance, and ocular surface pathology, andremains an unmet need in ocular disease with limited effectivetherapeutic options available. Dry eye is a major public health concernin an aging population, affecting up to one-third of the globalpopulation, including 5 million Americans aged 50 and over.Over-the-counter artificial tears and implantable punctal plugs arefrequently used for symptomatic relief. Therapeutic approaches involvereducing ocular surface inflammation or augmenting tear/mucin secretion.The only medication currently approved for dry eye is topicalcyclosporine, an anti-inflammatory that does not eliminate all symptomsin most dry eye patients. Accordingly, additional treatments are neededfor moderate-to-severe dry eye. Described herein, inter alia, aresolutions to these and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compounds having the formula:

In the compound of formula I, X is a bond, —O—, —N(R¹⁰)— (e.g. —NH—), or—S—. In embodiments, X is —O—, —N(R¹⁰)— (e.g. NH), — or —S—. Inembodiments, X is —O— or —S—. R¹ is hydrogen, halogen, —CX^(1.1) ₃,—CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C),—NHNR^(1B)R^(1C), —ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C),—NHC(O)NR^(1B)R^(1C), —N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D),—C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² ishydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A),substituted or unsubstituted alkyl (e.g. haloalkyl), substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R² is hydrogen, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃,—OR^(2A), substituted or unsubstituted alkyl (e.g. —CH₃, C₂-C₈ alkyl, orC₂-C₄ alkyl), substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl. R³ is hydrogen, —C(O)R^(3D), —C(O)NHNR^(3B)R^(3C),—C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl. In embodiment, R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl. R⁴ ishydrogen, —C(O)R^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R⁴ is hydrogen, —C(O)R^(4D), —C(O)NHNR^(4B)R^(4C),—C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. R³ and R⁴may optionally be joined to form, together with the atoms to which theyare attached, a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl. R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl. R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO₆R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO₇R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C), —ONR^(7B)R^(7C),—NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C), —N(O)_(m7),—NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A),—NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7C)(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In embodiments, R⁷ is hydrogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO₇R^(7A), —SO_(v7)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7A)C(O)R^(7C), —NR^(7C)(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R¹ ishydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(1.1), —CN,—SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C), —NHNR^(8B)R^(8C),—ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C),—N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(1D),—C(O)NR^(8B)R^(8C), —OR^(A), —NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D),—NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiments, R¹ is hydrogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(BA),—SO_(v8)NR^(8B)R^(8C), —NHNR^(1B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m8),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R¹ andR⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ are optionally joined to form,together with the atoms to which they are attached, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R¹⁰ is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.R^(1A), R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C), R^(2D), R^(3A),R^(3B), R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B),R^(5C), R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C),R^(7D), R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) andR^(9D) are independently hydrogen, halogen, —CF₃—CCl₃, —CBr₃, —CI₃, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B),R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C),R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to the samenitrogen atom may optionally be joined to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl. X^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F. The symbols n1, n2, n6, n7, n8, and n9are independently an integer from 0 to 4. The symbols m1, m6, m7, m8,m9, v1, v6, v7, v8 and v9 each independently 1 or 2. In embodiments,when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) isfluorine; R³ is hydrogen; and R¹ is R⁴ is —CH₃, then R⁶ is not —NO₂. Inembodiments, when X is —O—; R² is —(CH₂)_(n2)CX^(21′)3; n2 is 1; X^(2.1)is fluorine; R³ is hydrogen; and R¹ is substituted or unsubstitutedC₁-C₃ alkyl, then R⁹ is not —NO₂. In embodiments, when X is —O—; R² is—CH₂(CF₂)₂H; R³ is hydrogen; and R is R is —CH₃, then R¹ is not —NO₂. Inembodiments, when X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ are independentlyunsubstituted C₁-C₃ alkyl, then R¹ is not hydrogen. In embodiments, whenX is —O— and R² is methyl substituted with cycloalkyl, then R³ and R⁴are hydrogen.

Also provided herein are pharmaceutical compositions. In one aspect is apharmaceutical composition that includes a compound described herein orpharmaceutically acceptable salt thereof (e.g. a compound of formula I).

Further provided herein are methods of activating a Cystic FibrosisTransmembrane Conductance Regulator (CFTR). The method includescontacting the CFTR with an effective amount of a compound describedherein, thereby activating the CFTR.

Further provided herein are methods of treating a disease or disorder ina subject in need thereof by administering to said subject an effectiveamount of a compound described herein (e.g. a compound of formula I).

Further provided herein are methods of treating a disease or disorder ina subject in need thereof by administering to said subject an effectiveamount of a compound as described herein (e.g. a compound of formula I).In one aspect is a method of treating constipation in a subject in needthereof, the method including administering to the subject an effectiveamount described compound as described herein (e.g. a compound offormula I). In another aspect, is a method of treating a dry eyedisorder in a subject in need thereof, the method includingadministering to the subject an effective amount of a compound asdescribed herein (e.g. a compound of formula I). In yet another aspect,is a method of increasing lacrimation in a subject in need thereof, themethod including administering to the subject an effective amount acompound as described herein (e.g. a compound of formula I).

In one aspect, provided is a method of treating a cholestatic liverdisease in a subject in need thereof, including administering to thesubject an effective amount a compound as described herein (e.g. acompound of formula I). In another aspect, provided is a method oftreating a pulmonary disease or disorder in a subject in need thereof,including administering to the subject an effective amount of asdescribed herein (e.g. a compound of formula I). In embodiments, thepulmonary disease or disorder is chronic obstructive pulmonary disease(e.g. bronchitis, asthma, cigarette smoke-induced lung dysfunction).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Strategy for pre-clinical development of CFTR activators for dryeye therapy. Activators of human wild-type CFTR activators identified byhigh-throughput screening are confirmed and characterized using byelectrophysiological and biochemical assays, and then tested in livemice for activity at the ocular surface by measurements of potentialdifference and tear fluid secretion. The best compounds are then testedfor pharmacokinetic properties and efficacy in a dry eye rodent model.

FIGS. 2A-2D. In vitro characterization of CFTR activators. FIG. 2A)(Top) Chemical structures. (Bottom) Representative short-circuit current(I_(sc)) measured in Fischer rat thyroid (FRT) cells expressingwild-type CFTR. CFTR current was stimulated by test compounds andforskolin, and inhibited by CFTR_(inh)-172 (10 μM). FIG. 2B)Concentration-dependence of CFTR activators (each data set derived froma single dose-response experiment as in A and fitted using anexponential curve). One-hundred percent CFTR activation is defined asthat produced by 20 μM forskolin. FIG. 2C) I_(sc) measurement for VX-770done as in A. FIG. 2D) Cellular cAMP concentration in FRT cells inresponse to incubation for 10 min with 5 μM test compounds without orwith forskolin (fsk, 100 nM). Positive controls included forskolin (100nM and 20 μM), and forskolin plus 3-isobutyl-1-methylxanthine (IBMX, 100μM) (mean±SEM, n=4-8).

FIGS. 3A-3E. Potential difference (PD) measurements of CFTR activatorsat the ocular surface in live mice. FIG. 3A) (Left) Photograph of ananesthetized mouse demonstrating ocular surface perfusion for PDmeasurement. The perfusion catheter, attached to the measuringelectrode, is oriented perpendicular to the ocular surface.Cross-clamping forceps retract the upper eyelid to expose cornea andbulbar/palbebral conjunctiva for perfusion. The reference electrode isgrounded via subcutaneous butterfly needle. (Right) Schematic of PDtracing for a typical experiment testing CFTR activity, as described inResults. FIG. 3B) Representative ocular surface PD measurements inwild-type mice. Solution compositions are detailed in Ref. 22.Concentrations: amiloride, 100 μM; forskolin and CFTR_(inh)-172, 10 μM;test compounds, 1-10 μM as indicated. FIG. 3C) Study as in C, but withVX-770, 1-10 μM, as indicated. FIG. 3D) Summary of ΔPD in wild-type miceproduced by forskolin (20 μM), or test compounds or VX-770 (each 1 μM).PDs were recorded in the presence of 100 μM amiloride and in thepresence of an outward apical Cl⁻ gradient (mean±SEM, 8-20 eyes peragonist tested). FIG. 3E) Representative ocular surface PD measurementsin CF mouse. Study as in B & C, CFTR_(act)-K032, 1-10 μM as indicated.

FIGS. 4A-4D. Tear fluid secretion measurement of CFTR activators inliving mice. FIG. 4A) Tear fluid was measured just prior to and atindicated times after single-dose topical application of vehicle (PBS,0.5% polysorbate, 0.5% DMSO), cholera toxin (0.1 μg/mL), forskolin (20μM), or forskolin+IBMX (250 μM). The effect of cholera toxin wasmeasured after pre-anesthetizing the ocular surface with 4% lidocaine tosuppress irritation and reflex tear secretion (mean±SEM, 6-10 eyes percondition). FIG. 4B) Time course of tear secretion following topicaldelivery of indicated compound. Concentrations: CFTR_(act)-B074, 100 μM;CFTR_(act)-J027, 50 μM; CFTR_(act)-K089, 50 μM; VX-770, 10 μM (mean±SEM,6-18 eyes). FIG. 4C) Effect of repeated dosing. CFTR_(act)-J027 (0.1nmol) was topically applied three times a day for two days. Tear fluidmeasurements were done after Dose 1 and Dose 2 on day 1, and Dose 5 onday 2 (mean±SEM, n=6 eyes). FIG. 4D) Lack of effect of CFTR activatorson tear fluid secretion in CF mice, with compounds tested at the sameconcentrations as in B.

FIGS. 5A-5C. Compound pharmacology. FIG. 5A) Liquid chromatography/massspectroscopy (LC/MS) determination of CFTR_(act)-K089 amount in tearfluid at indicated times following single-dose (0.1 nmol)administration. Representative background-subtracted peak areas fromtear washes (left) and means of corresponding amount recovered (right)(mean±SEM, 4 eyes per time point). Dashed lines denote the upper andlower calculated quantities of CFTR_(act)-K089 required to achieve EC₅₀concentration. FIG. 5B) Lissamine green staining of comea in BALB/cmice, measured on a 12-point scale (see Methods) after 14-days of threetimes daily treatment with CFTR activators (0.1 nmol) or vehicle(mean±SEM, 6 eyes per group). Shown as a positive control are scoresfrom vehicle-treated mice following lacrimal gland excision (LGE) on Day0 (n=11 eyes; *P<0.001 compared with other groups). FIG. 5C)Cytotoxicity measured by Alamar Blue assay in FRT cells incubated withtest compounds for 1 or 24 h (10% DMSO as positive control; *P<0.05compared to untreated cells; P=0.02 and 0.0006 for 1 and 24 h,respectively) (mean±SEM, n=4).

FIGS. 6A-6C. Topical CFTR_(act)-K089 restores tear secretion andprevents corneal epithelial disruption following LGE. FIG. 6A) Basaltear secretion following extraorbital LGE in BALB/c mice, comparing eyestreated with CFTR_(act)-K089 (mean±SEM, 15 eyes) to vehicle (n=1 eyes).Tear volume was measured immediately prior to LGE, and then one hourafter the first daily dose on Days 4, 10 and 14 after LGE. *P<0.001.FIG. 6B) Representative photographs of eyes prior to LGE (left) and onDay 14 after LGE (right) in vehicle-treated eyes (top) andCFTR_(act)-K089-treated eyes (bottom). FIG. 6C) Corneal epithelialdisruption after LGE measured by LG scoring on a 12-point scale in thesame eyes as in A (mean±SEM). *P<0.001.

FIG. 7. A summary of EC₅₀ and V_(max) values for compounds screenedagainst CFTR A cell-based functional high-throughput screen of 120,000compounds at 10 μM identified 20 chemical classes of small-moleculeactivators of wild-type CFTR that produced >95% of maximal CFTRactivation. The screen was done in FRT epithelial cells co-expressinghuman wild-type CFTR and a cytoplasmic YFP halide sensor in 96-wellformat (26, 31, 32). Details of the primary screen will be reportedseparately. Secondary screening involved I_(sc) measurement inCFTR-expressing FRT cells pretreated with submaximal forskolin (50 nM).Twenty-one compounds from eight chemical classes produced largeincreases in I_(sc) at 1 μM (>75% of maximal current produced by 20 μMforskolin).

FIGS. 8A-8D. Identification of small-molecule CFTR activators. FIG. 8A.Project overview. FIG. 8B. CFTR activator screen using FRT cellscoexpressing human wild-type CFTR and YFP iodide-sensing protein. Testcompounds at 10 μM were added for 10 min at room temperature in thepresence of forskolin (125 nM) before iodide addition. Examples of datafrom single wells of a 96-well plate showing CFTR activation byCFTR_(act)-J027. FIG. 8C. Structures of CFTR activators emerging fromthe screen. FIG. 8D. Synthesis of CFTR_(act)-J027.

FIGS. 9A-9E. Characterization of CFTR activation by CFTR_(act)-J027.Short-circuit current measured in FRT cells expressing human wild-typeCFTR (FIG. 9A) and ΔF508-CFTR (FIG. 9C) showing responses to indicatedconcentrations of forskolin (fsk), CFTR_(act)-J027, and VX-770. TheΔF508-CFTR-expressing FRT cells were corrected with 3 μM VX-809 at 37°C. for 24 h before measurement CFTR_(inh)-172 (Inh-172, 10 μM) was addedwhere indicated. FIG. 9B. CFTR_(act)-J027 concentration-dependentactivation of wild-type CFTR Cl⁻ current (S.E.; n=3 cultures). FIG. 9D.Short-circuit current in mouse colon showing responses to indicatedconcentrations of forskolin (fsk), CFTR_(act)-J027, and CFTR_(inh)-172.FIG. 9E. Assay of cAMP concentration in FRT cells measured following10-min incubation with indicated concentrations of forskolin and 5 μMCFTR_(act)-J027. Positive controls included forskolin (100 nM and 20μM), and forskolin plus 3-isobutyl-1-methylxanthine (IBMX, 100 μM)(mean±SE, n=4-8).

FIGS. 10A-10D. CFTR_(act)-J027 normalizes stool output and water contentin loperamide-treated mice. FIG. 10A. Mouse model of constipation withloperamide (left). Three-hour stool weight, number of pellets, and stoolwater content in mice (mean±S.E., 6 mice per group). FIG. 10B. Samestudy as in A, but with cystic fibrosis mice lacking function CFTR (3-6mice per group). FIG. 10C. Same study in A, but with an inactivechemical analog of CFTR_(act)-J027 (structure shown). FIG. 10D.Dose-response for intraperitoneal administration of CFTR_(act)-J027 inloperamide-treated mice (4-6 mice per group). One-way analysis ofvariance was used for A and B, Student's t-test was used for C, *p<0.05,***p<0.001, ns: not significant.

FIGS. 11A-11C. Orally administered CFTR_(act)-J027 normalizes stooloutput and water content in loperamide-treated mice. FIG. 11A. Studyprotocol (left) and stool output, pellet number and water content asdone in FIG. 3 (mean±S.E., 6 mice per group). FIG. 11B. Dose-responsestudy of CFTR_(act)-J027 administered orally in loperamide-treated mice(4-6 mice per group). FIG. 11C. Same study in FIG. 11A, but with orallubiprostone (0.5 mg/kg) or linaclotide (0.5 mg/kg) (5-6 mice pergroup). One-way analysis of variance, *p<0.05, **p<0.01, ***p<0.001, ns:not significant.

FIGS. 12A-12D. CFTR_(act)-J027 actions on intestinal fluid secretion,absorption and motility. FIG. 12A. Whole-gut transit time in control andloperamide-treated wild-type (left) and cystic fibrosis (right) mice(mean±S.E., 3-5 mice per group). Where indicated loperamide (0.3 mg/kg)and CFTR_(act)-J027 (10 mg/kg) was administered intraperitoneally at 0time (mean±S.E., 6 mice per group). One-way analysis of variance,**p<0.01, ***p<0.001, ns: not significant. FIG. 12B. Contraction ofisolated intestinal strips. Ileum and colon strips (˜2 cm) weresuspended in Krebs-Henseleit buffer with 0.5 g and 0.2 g tension,respectively. Where indicated CFTR_(act)-J027, loperamide and carbacholwere added to the organ chamber. FIG. 12C. Intestinal fluid secretionmeasured in closed mid-jejunal loops in wild-type mice (upper panel).Loops were injected with 100 μL vehicle or 100 gig CFTR_(act)-J027. Loopweight/length was measured at 90 min (mean±S.E., 4 loops per group).Similar experiments done in cystic fibrosis mice (lower panel). FIG.12D. Intestinal fluid absorption measured in mid-jejunal loops in cysticfibrosis mice. Loops were injected with 100 μL vehicle or 0.1 mgCFTR_(act)-J027. Loop weight/length was measured at 30 min. Summary offluid absorption (mean±S.E., 4 loops per group). Student's t-test,**p<0.01, ***p<0.001, ns: not significant.

FIGS. 13A-13E. FIG. 13A. In vitro metabolic stability of CFTR_(act)-J027assayed in mouse liver microsomes after incubation for specified times.FIG. 13B. Standard plasma concentration curve for LC-MS (left) andkinetics of CFTR_(act)-J027 concentration in plasma determined by LC/MSfollowing bolus intraperitoneal or oral administration of 10 mg/kgCFTR_(act)-J027 at zero time (right, mean±S.E., 3 mice per group). FIG.13C. In vitro toxicity measured by Alamar Blue assay in FRT cells. FIG.13D. Body weight and lung wet/dry weight ratio in mice receiving 10mg/kg CFTR_(act)-J027 orally for 7 days (mean±S.E., 5 mice per group).FIG. 13E. Chronic administration protocol (left) and efficacy of oralCFTR_(act)-J027 after 7-day administration (mean±S.E., 5 mice pergroup). Student's t-test, *p<0.05, **p<0.01, ***p<0.001, ns: notsignificant.

FIGS. 14A-14B. Structure-activity analysis of aminophenyl-1,3,5-triazineCFTR activators. FIG. 14A. Chemical structure of CFTR_(act)-K089 (1).FIG. 14B. Preliminary SAR analysis based on commercial analogs (seeTable 2 for data on all commercial analogs).

FIGS. 15A-15B. Short-circuit current measurement of CFTR activation by6k and 12. FIG. 15A. Measurements done in FRT cells expressing humanwildtype CFTR showing responses to indicated concentrations offorskolin, 6k or 12, and 10 μM CFTR inhibitor CFTR_(inh)-172. FIG. 15B.Concentration-dependent activation of CFTR (mean±S.E.M., n=3).

FIGS. 16A-16E. Characterization of 6k and 12. FIG. 16A. Cellular cAMP inFRT cells following incubation for 10 min with 10 μM 6k or 12, withoutor with 90 nM forskolin (fsk), as well as forskolin alone (90 nM and 20μM) and forskolin (20 μM)+IBMX (100 μM) (mean±S.E.M., n=4). FIG. 16B.Cytoplasmic calcium measured by Fluo-4 fluorescence. FRT cells werepretreated for 5 min with 10 μM 6k or 12 (or control), with 100 μM ATPadded as a calcium agonist as indicated. FIG. 16C. TMEM16A activitymeasured in FRT cells expressing YFP showing no inhibition (left,iodide+ATP addition) or activation (right, iodide addition) by 10 μM 6kor 12. FIG. 16D. CaCC activity measured in HT-29 cells expressing YFPshowing no activation (iodide addition) or inhibition (iodide+ATPaddition) by 10 μM 6k or 12. FIG. 16E. (left) Short circuit-current inprimary cultures of human bronchial epithelial cells in response toagonists and inhibitors that target key ion transport processes: 20 μMamiloride (ami); 20 μM forskolin (fsk); 10 μM CFTR_(inh)-172; 100 μMATP. Experiments were done without activator or following 10-minpreincubation with 10 μM 6k or 12 (right). CFTR activation by 12 after100 nM forskolin (20 μM amil, 10 μM CFTR_(inh)-172, 100 μM ATP). Studiesin B-E are representative of 2-4 separate sets of experiments.

FIGS. 17A-17B. Compound pharmacology. FIG. 17A. Cytotoxicity wasmeasured by Alamar Blue assay in FRT cells incubated for 8 h with 10 μM6k or 12, with 33% DMSO as positive control (mean±S.E.M., n=8). FIG.17B. In vitro metabolic stability. Compounds at 5 μM were incubated forindicated times with 1 mg/ml hepatic microsomes in the presence of NADPHand parent compound assayed by LC/MS (mean±S.E.M., n=3). LC/MS profileof 12 shown on the right with elution time on the x-axis for incubationtimes of 0, 15 and 60 min.

FIGS. 18A-18C. Tear fluid volume in mice following ocular delivery of 1or 12. FIG. 18A. Tear volume was measured just before and at theindicated times after single ocular delivery of vehicle, 1 (250 pmol) or12 (250 pmol) in a 2.5 μL volume. FIG. 18B. Study as in A but in CF micelacking functional CFTR. FIG. 18C. Single dose study as in A withdifferent amounts of 12. Data reported as mean±S.E.M, 5 mice, 10 eyesper condition, * p<0.05, ** p<0.01 compared to vehicle control.

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH2O— is equivalent to —OCH2-.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain.Examples of saturated hydrocarbon radicals include, but are not limitedto, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., selected from the group consisting of O, N, P, Si,and S), and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) (e.g., O, N, P, S, B, As, and Si) may be placed at anyinterior position of the heteroalkyl group or at the position at whichthe alkyl group is attached to the remainder of the molecule.Heteroalkyl is an uncyclized chain. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheteroalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl,pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl,oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl,indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl,quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be a —O— bonded to a ring heteroatomnitrogen.

A “used ring aryl-heterocycloalkyl” is an aryl fused to aheterocycloalkyl. A “used ring heteroaryl-heterocycloalkyl” is aheteroaryl fused to a heterocycloalkyl. A “fused ringheterocycloalkyl-cycloalkyl” is a heterocycloalkyl fused to acycloalkyl. A “fused ring heterocycloalkyl-heterocycloalkyl” is aheterocycloalkyl fused to another heterocycloalkyl. Fused ringaryl-heterocycloalkyl, fused ring heteroaryl-heterocycloalkyl, fusedring heterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substituentsdescribed herein. Fused ring aryl-heterocycloalkyl, fused ringheteroaryl-heterocycloalkyl, fused ring heterocycloalkyl-cycloalkyl, orfused ring heterocycloalkyl-heterocycloalkyl may each independently benamed according to the size of each of the fused rings. Thus, forexample, 6,5 aryl-heterocycloalkyl fused ring describes a 6 memberedaryl moiety fused to a 5 membered heterocycloalkyl. Spirocyclic ringsare two or more rings wherein adjacent rings are attached through asingle atom. The individual rings within spirocyclic rings may beidentical or different Individual rings in spirocyclic rings may besubstituted or unsubstituted and may have different substituents fromother individual rings within a set of spirocyclic rings. Possiblesubstituents for individual rings within spirocyclic rings are thepossible substituents for the same ring when not part of spirocyclicrings (e.g. substituents for cycloalkyl or heterocycloalkyl rings).Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkylene andindividual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.all rings being substituted heterocycloalkylene wherein each ring may bethe same or different substituted heterocycloalkylene). When referringto a spirocyclic ring system, heterocyclic spirocyclic rings means aspirocyclic rings wherein at least one ring is a heterocyclic ring andwherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R′″, —CN, —NO₂, —NR′SO₂R″, —NR′C═(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2 m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR″″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C═(O)R″, —NR′C(O)—OR″, —NR′OR″,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), Boron(B), Arsenic (As), and silicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

(A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl, and(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:(i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl, and(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:(a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstitutedheteroaryl, and(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from:oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,—NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstitutedheteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or“lower substituent group,” as used herein, meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

Certain compounds described herein possess asymmetric carbon atoms(optical or chiral centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the (R) and (S)configurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds, generally recognized as stable bythose skilled in the art, are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, replacement of fluoride by ¹⁸F, or the replacement of a carbonby ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), fluoride (¹⁸F), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). Allisotopic variations of the compounds of the present invention, whetherradioactive or not, are encompassed within the scope of the presentinvention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

Where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I), orFormula I), a Roman decimal symbol may be used to distinguish eachappearance of that particular R group. For example, where multiple R¹³substituents are present, each R¹³ substituent may be distinguished asR^(13.1), R^(13.2), R^(13.3), R^(13.4), etc., wherein each of R^(13.1),R^(13.2), R^(13.1), R^(13.4), etc. is defined within the scope of thedefinition of R¹³ and optionally differently. The terms “a” or “an,” asused in herein means one or more. In addition, the phrase “substitutedwith a[n],” as used herein, means the specified group may be substitutedwith one or more of any or all of the named substituents. For example,where a group, such as an alkyl or heteroaryl group, is “substitutedwith an unsubstituted C₁-C₂₀ alkyl, or unsubstituted 2 to 20 memberedheteroalkyl,” the group may contain one or more unsubstituted C₁-C₂₀alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.

Description of compounds of the present invention is limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

“Analog,” or “analogue” are used in accordance with plain ordinarymeaning within Chemistry and Biology and refer to a chemical compoundthat is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analogue is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The terms “cystic fibrosis transmembrane conductance regulator,” and“CFTR” are here used interchangeably and according to their common,ordinary meaning and refer to proteins of the same or similar names andfunctional fragments and homologs thereof. The term includes anyrecombinant or naturally occurring form of, or variants thereof thatmaintain CFTR activity (e.g. within at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100% activity compared to CFTR).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereininclude those compounds that readily undergo chemical or enzymaticchanges under physiological conditions to provide the compounds of thepresent invention. Additionally, prodrugs can be converted to thecompounds of the present invention by chemical or biochemical methods inan ex vivo environment. For example, prodrugs can be slowly converted tothe compounds of the present invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

The terms “treating”, or “treatment” refer to any indicia of success inthe treatment or amelioration of an injury, disease, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; or improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters, including the results of a physicalexamination, neuropsychiatric exams, and/or a psychiatric evaluation.The term “treating” and conjugations thereof, include prevention of aninjury, pathology, condition, or disease.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, increase enzyme activity, reduce one ormore symptoms of a disease or condition). An example of an “effectiveamount” is an amount sufficient to contribute to the treatment,prevention, or reduction of a symptom or symptoms of a disease, whichcould also be referred to as a “therapeutically effective amount.” A“reduction” of a symptom or symptoms (and grammatical equivalents ofthis phrase) means decreasing of the severity or frequency of thesymptom(s), or elimination of the symptom(s). A “prophylacticallyeffective amount” of a drug is an amount of a drug that, whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset (or reoccurrence) of an injury,disease, pathology or condition, or reducing the likelihood of the onset(or reoccurrence) of an injury, disease, pathology, or condition, ortheir symptoms. The full prophylactic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations. The exact amounts willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Pickar, DosageCalculations (1999); and Remington: The Science and Practice ofPharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams &Wilkins).

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In embodiments, a controlis the measurement of the activity of a protein in the absence of acompound as described herein (including embodiments and examples).

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be a compoundas described herein and a protein or enzyme. Contacting may includeallowing a compound described herein to interact with a protein orenzyme that is involved in a signaling pathway.

As defined herein, the term “activation,” “activate,” “activating” andthe like in reference to a protein-activator interaction meanspositively affecting (e.g. increasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the activator. Activation may refer to reduction of a diseaseor symptoms of disease. Activation may refer to an increase in theactivity of a particular protein or nucleic acid target. The protein maybe cystic fibrosis transmembrane conductance regulator. Thus, activationincludes, at least in part, partially or totally increasing stimulation,increasing, promoting, or expediting activation, or activating,sensitizing, or up-regulating signal transduction or enzymatic activityor the amount of a protein.

The term “modulator” refers to a composition that increases or decreasesthe level of a target molecule or the function of a target molecule orthe physical state of the target of the molecule.

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, a modulator of a target proteinchanges by increasing or decreasing a property or function of the targetmolecule or the amount of the target molecule. A modulator of a diseasedecreases a symptom, cause, or characteristic of the targeted disease.

“Selective” or “selectivity” or the like of a compound refers to thecompound's ability to discriminate between molecular targets.“Specific”, “specifically”, “specificity”, or the like of a compoundrefers to the compound's ability to cause a particular action, such asinhibition, to a particular molecular target with minimal or no actionto other proteins in the cell.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The compositions disclosed herein can be delivered by transdermally, bya topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols. Oral preparations include tablets, pills,powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups,slurries, suspensions, etc., suitable for ingestion by the patient.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions. The compositions of thepresent invention may additionally include components to providesustained release and/or comfort. Such components include high molecularweight, anionic mucomimetic polymers, gelling polysaccharides andfinely-divided drug carrier substrates. These components are discussedin greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and4,861,760. The entire contents of these patents are incorporated hereinby reference in their entirety for all purposes. The compositionsdisclosed herein can also be delivered as microspheres for slow releasein the body. For example, microspheres can be administered viaintradermal injection of drug-containing microspheres, which slowlyrelease subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645,1995; as biodegradable and injectable gel formulations (see, e.g., GaoPharm. Res. 12:857-863, 1995); or, as microspheres for oraladministration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674,1997). In another embodiment, the formulations of the compositions ofthe present invention can be delivered by the use of liposomes whichfuse with the cellular membrane or are endocytosed, i.e., by employingreceptor ligands attached to the liposome, that bind to surface membraneprotein receptors of the cell resulting in endocytosis. By usingliposomes, particularly where the liposome surface carries receptorligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of thecompositions of the present invention into the target cells in vivo.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn,Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989). The compositions can also be delivered asnanoparticles.

Pharmaceutical compositions may include compositions wherein the activeingredient (e.g. compounds described herein, including embodiments orexamples) is contained in a therapeutically effective amount, i.e., inan amount effective to achieve its intended purpose. The actual amounteffective for a particular application will depend, inter alia, on thecondition being treated. When administered in methods to treat adisease, such compositions will contain an amount of active ingredienteffective to achieve the desired result, e.g., modulating the activityof a target molecule, and/or reducing, eliminating, or slowing theprogression of disease symptoms.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and compounds of Applicants' invention. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art.

The compounds described herein can be used in combination with oneanother, with other active drugs known to be useful in treating adisease (e.g. anticonstipation, anti-dry eye, anti-pulmonary disease,anti-liver disease, or anti-lung disease) or with adjunctive agents thatmay not be effective alone, but may contribute to the efficacy of theactive agent. Thus, the compounds described herein may beco-administered with one another or with other active drugs known to beuseful in treating a disease.

By “co-administer” it is meant that a compound described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapies, for example, ananti-constipation or anti-dry eye agent as described herein. Thecompounds described herein can be administered alone or can beco-administered to the patient. Co-administration is meant to includesimultaneous or sequential administration of the compound individuallyor in combination (more than one compound or agent). Thus, thepreparations can also be combined, when desired, with other activesubstances (e.g. anti-constipation or anti-dry eye agents).

Co-administration includes administering one active agent (e.g. acomplex described herein) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or24 hours of a second active agent (e.g. anti-constipation or anti-dryeye agents). Also contemplated herein, are embodiments, whereco-administration includes administering one active agent within 0.5, 1,2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a second active agent.Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1, 5,10, 15, 20, or 30 minutes of each other), or sequentially in any order.Co-administration can be accomplished by co-formulation, i.e., preparinga single pharmaceutical composition including both active agents. Inother embodiments, the active agents can be formulated separately. Theactive and/or adjunctive agents may be linked or conjugated to oneanother. The compounds described herein may be combined with treatmentsfor constipation and dry eye disorders.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease means thatthe disease is caused by (in whole or in part), a symptom of the diseaseis caused by (in whole or in part) the substance or substance activityor function, or a side-effect of the compound (e.g. toxicity) is causedby (in whole or in part) the substance or substance activity orfunction.

“Patient,” “subject,” “patient in need thereof,” and “subject in needthereof” are herein used interchangeably and refer to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In some embodiments, a patient is human.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with the compounds ormethods provided herein. Disease as used herein may refer toconstipation or dry eye disorders.

Examples of anti-constipation agents include, but are not limited todiphenylmethanes, Lactobacillus paracasei, linaclotide and lubiprostone.Examples of anti-dry eye agents include, but are not limited to, topicalcyclosporine, P321 (an ENaC inhibitor) and Diquafosol.

I. Compositions

Provided herein are compounds having the formula:

In the compound of formula I, X is a bond, —O—, —N(R¹⁰)— (e.g. —NH—), or—S—. In embodiments, X is —O—, —N(R¹⁰)— (e.g. NH), — or —S—. Inembodiments, X is —O— or —S—. R¹ is hydrogen, halogen, —CX^(1.1) ₃,—CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C),—NHNR^(1B)R^(1C), —ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C),—NHC(O)NR^(1B)R^(1C), —N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D),—C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R² ishydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A),substituted or unsubstituted alkyl (e.g. haloalkyl), substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R² is hydrogen, —CH₃, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃,—OR^(2A), substituted or unsubstituted C₂-C₈ alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl. In embodiments, R² is hydrogen, —CX^(2.1) ₃, CHX^(2.1) ₂,—(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted alkyl(e.g. C₂-C₄ haloalkyl), substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl. In embodiments, R² is hydrogen, —CH₃,—CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted C₂-C₈ alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl. R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl. In embodiment, R³ is hydrogen,—C(O)R^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A),—C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl. R⁴ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A)C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl. In embodiments, R⁴ is hydrogen,—C(O)R^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl. R³ and R⁴ may optionally be joined to form,together with the atoms to which they are attached, a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl. R⁵ is hydrogen, —C(O)R^(5D), —C(O)NHNR^(5B)R^(5C),—C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl. R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —SO₆R^(6A), —SO_(v6)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D), —C(O)OR^(6D),—C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A), —NR^(6B)C(O)R^(6D),—NR⁶C(O)OR⁶, —NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁷ is hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R⁷ is hydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1),—CN, —SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D),—C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C),—NR^(7B)C(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R¹ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A),—SO_(v8)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m8),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(1D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In embodiments, R¹ is hydrogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)R^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O),—NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(1D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(8D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹are optionally joined to form, together with the atoms to which they areattached, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R¹⁰ is hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R^(1A), R^(1B), R^(1C), R^(1D), R^(2A),R^(2B), R^(2C), R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B),R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C),R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D),R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen, halogen,—CF₃—CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) andR^(9C) substituents bonded to the same nitrogen atom may optionally bejoined to form, together with the atoms to which they are attached, asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl. X^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1)and X^(9.1) are independently —Cl, —Br, —I or —F. n1, n2, n6, n7, n8,and n9 are independently an integer from 0 to 4. m1, m6, m7, m8, m9, v1,v6, v7, v8 and v9 each independently 1 or 2.

In embodiments, n1 is 0. In embodiments, n1 is 1. In embodiments, n1 is2. In embodiments, n1 is 3. In embodiments, n1 is 4. In embodiments, n2is 0. In embodiments, n2 is 1. In embodiments, n2 is 2. In embodiments,n2 is 3. In embodiments, n2 is 4. In embodiments, n6 is 0. Inembodiments, n6 is 1. In embodiments, n6 is 2. In embodiments, n6 is 3.In embodiments, n6 is 4. In embodiments, n7 is 0. In embodiments, n7is 1. In embodiments, n7 is 2. In embodiments, n7 is 3. In embodiments,n7 is 4. In embodiments, n8 is 0. In embodiments, n8 is 1. Inembodiments, n8 is 2. In embodiments, n8 is 3. In embodiments, n8 is 4.In embodiments, n9 is 0. In embodiments, n9 is 1. In embodiments, n9 is2. In embodiments, n9 is 3. In embodiments, n9 is 4. In embodiments, m1is 1. In embodiments, m1 is 2. In embodiments, m6 is 1. In embodiments,m6 is 2. In embodiments, m7 is 1. In embodiments, m7 is 2. Inembodiments, m8 is 1. In embodiments, m8 is 2. In embodiments, m9 is 1.In embodiments, m9 is 2. In embodiments, v1 is 1. In embodiments, v1 is2. In embodiments, v6 is 1. In embodiments, v6 is 2. In embodiments, v7is 1. In embodiments, v7 is 2. In embodiments, v8 is 1. In embodiments,v8 is 2. In embodiments, v9 is 1. In embodiments, v9 is 2.

In embodiments, when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R¹ is substituted orunsubstituted C₁-C₃ alkyl, then R⁶ is not —N(O)_(m6). In embodiments,when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) isfluorine; R³ is hydrogen; and R¹ is substituted or unsubstituted C₁-C₃alkyl, then R⁹ is not —N(O)_(m9). In embodiments, when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is —CH₃, then R⁶ is not —NO₂. In embodiments, when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is —CH₃, then R⁹ is not —NO₂. In embodiments, when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is substituted or unsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂. Inembodiments, when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X²¹ isfluorine; R³ is hydrogen; and R¹ is substituted or unsubstituted C₁-C₃alkyl, then R⁹ is not —NO₂. In embodiments, when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is —CH₃, then R⁶ is not —NO₂. In embodiments, when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is —CH₃, then R⁹ is not —NO₂. In embodiments, when X is —O—; R² is—CH₂CF₃; one of R³ and R is hydrogen and one of R³ and R is —CH₃, thenR⁶ is not —NO₂. In embodiments, when X is —O—; R² is —CH₂CF₃; one of R³and R is hydrogen and one of R³ and R is ethyl, then R⁶ is not —NO₂. Inembodiments, when X is —O—; R² is —CH₂CF₃; one of R³ and R is hydrogenand one of R³ and R is unsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂.In embodiments, when X is —O—; R² is —CH₂CF₃; one of R³ and R isunsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂. In embodiments, when Xis —O—; R² is —CH₂CF₃; R³ and R⁴ are hydrogen or unsubstituted C₁-C₃alkyl, then R⁶ is not —NO₂. In embodiments, when X is —O—; R² is—CH₂CF₃; one of R³ and R is hydrogen and one of R³ and R is —CH₃, thenR⁹ is not —NO₂. In embodiments, when X is —O—; R² is —CH₂CF₃; one of R³and R is hydrogen and one of R³ and R is ethyl, then R⁹ is not —NO₂. Inembodiments, when X is —O—; R² is —CH₂CF₃; one of R³ and R⁴ is hydrogenand one of R³ and R⁴ is unsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂.In embodiments, when X is —O—; R² is —CH₂CF₃; one of R³ and R⁴ isunsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂. In embodiments, when Xis —O—; R² is —CH₂CF₃; R³ and R⁴ are hydrogen or unsubstituted C₁-C₃alkyl, then R⁹ is not —NO₂.

In embodiments, when X is —O—; R² is —CH₂(CF₂)₂H; R³ is hydrogen; and R¹is unsubstituted C₁-C₃ alkyl, then R¹ is not —N(O)_(m1). In embodiments,when X is —O—; R² is —CH₂(CF₂)₂H; R³ is hydrogen; and R¹ isunsubstituted C₁-C₃ alkyl, then R¹ is not —NO₂. In embodiments, when Xis —O—; R² is —CH₂(CF₂)₂H; one of R³ and R is hydrogen and one of R³ andR is —CH₃, then R¹ is not —NO₂. In embodiments, when X is —O—; R² is—CH₂(CF₂)₂H; one of R³ and R⁴ is hydrogen and one of R³ and R is ethyl,then R¹ is not —NO₂. In embodiments, when X is —O—; R² is —CH₂(CF₂)₂H;one of R³ and R is hydrogen and one of R³ and R is unsubstituted C₁-C₃alkyl, then R¹ is not —NO₂. In embodiments, when X is —O—; R² is—CH₂(CF₂)₂H; one of R³ and R is unsubstituted C₁-C₃ alkyl, then R¹ isnot —NO₂. In embodiments, when X is —O—; R² is —CH₂(CF₂)₂H; R³ and R⁴are hydrogen or —CH₃, then R¹ is not —NO₂. When X is —O—; R² is—CH₂(CF₂)₂H; R³ is hydrogen; and R¹ is substituted or unsubstitutedC₁-C₃ alkyl, then R¹ is not —NO₂. When X is —O—; R² is —CH₂(CF₂)₂H; R³is hydrogen; and R¹ is substituted or unsubstituted C₁-C₃ alkyl, then R¹is not —NO₂.

In embodiments, when X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ areindependently unsubstituted C₁-C₃ alkyl, then R¹ is not hydrogen. Inembodiments, when X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ are independentlymethyl or ethyl, then R¹ is not hydrogen. In embodiments, when X is —O—;R² is —CH(CF₃)₂; R³ and R⁴ are independently ethyl, then R¹ is nothydrogen. In embodiments, when X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ areindependently methyl, then R¹ is not hydrogen. In embodiments, when X is—O—; R² is —CH(CF₃)₂; one of R³ and R is methyl, one of R³ and R isethyl, then R¹ is not -hydrogen. In embodiments, when X is —O—; R² is—CH(CF₃)₂; R³ and R⁴ are independently C₁-C₃ alkyl, then R¹ is nothydrogen. When X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ are independentlysubstituted or unsubstituted C₁-C₃ alkyl, then R¹ is not hydrogen.

In embodiments, when X is —O— and R² is alkyl substituted withsubstituted or unsubstituted cycloalkyl, then R³ and R⁴ are hydrogen. Inembodiments, when X is —O— and R² is methyl substituted with substitutedor unsubstituted 5-7 membered cycloalkyl, then R³ and R⁴ are hydrogen.In embodiments, when X is —O— and R² is ethyl substituted with 5-7membered cycloalkyl, then R³ and R⁴ are hydrogen. In embodiments, when Xis —O— and R² is methyl substituted with substituted or unsubstitutedcyclohexyl, then R³ and R⁴ are hydrogen. In embodiments, when X is —O—and R² is methyl substituted with cyclohexyl, then R³ and R⁴ arehydrogen. In embodiments, when X is —O— and R² is C₁-C₃ alkylsubstituted with cyclohexyl, then R³ and R⁴ are hydrogen. Inembodiments, when X is —O— and R² is ethyl substituted with cyclohexyl,then R³ and R⁴ are hydrogen. In embodiments, when X is —O— and R² ismethyl substituted with substituted cyclohexyl, then R³ and R⁴ arehydrogen. In embodiments, when X is —O— and R² is ethyl substituted withsubstituted cyclohexyl, then R³ and R⁴ are hydrogen.

In embodiments, X is —O—, —NH— or —S—. In embodiments, X is —O— or —S—.In embodiments, X is —NH—. In embodiments, X is —O—.

In embodiments, R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂,—CH₂X^(1.1), —CN, —N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl. In embodiments, R¹ is hydrogen, halogen,—CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —N(O)_(m1), —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R¹ is hydrogen, halogen, —CF₃,—CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —N(O)_(m1), —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R¹ is hydrogen, halogen, —CX^(1.1)₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂, —NO, —C(O)R^(1A), —C(O)OR^(1D),—OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted or unsubstituted alkyl. Inembodiments, R^(1A) and R^(1D) are independently hydrogen or methyl. Inembodiments, R¹ is a hydrogen, halogen, —CN, —NO₂, —COH, —COCH₃,—COOCH₃, or —COOH. In embodiments, R¹ is halogen, —NO₂ or —COOH. Inembodiments, R¹ is halogen or —NO₂. In embodiments, R¹ is halogen or—COOH. In embodiments, R¹ is —NO₂. In embodiments, R¹ is a halogen.

In embodiments, R² is hydrogen, —CH₃, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1)₃, —OR^(2A), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R² is hydrogen, —CH₃, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃,—OR², substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, or substituted orunsubstituted heterocycloalkyl. In embodiments, R² is hydrogen, —CH₃,—CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl. Inembodiments, R² is hydrogen, —CH₃, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃,substituted or unsubstituted alkyl (e.g. haloalkyl). In embodiments, R²is hydrogen, —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, substituted C₂-C₈ alkyl(e.g. C₂-C₈ haloalkyl). In embodiments, R² is —CX^(2.1) ₃,—(CH₂)_(n2)CX^(2.1) ₃, substituted C₂-C₈ alkyl (e.g. C₂-C₈ haloalkyl).In embodiments, R² is CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, substitutedC₂-C₈ alkyl, or C₂-C₈₅ haloalkyl. In embodiments, R² is—(CH₂)_(n2)CX^(2.1) ₃, substituted C₂-C₈ alkyl (e.g. C₂-C₈ haloalkyl).In embodiments, R² is —(CH₂)_(n2)CX^(2.1) ₃ (e.g. C₂-C₅ haloalkyl). Inembodiments, R² is —CX^(2.1) ₃. In embodiments, R² is—(CH₂)_(n2)CX^(2.1) ₃. In embodiments, R² is C₂-C₈ alkyl substitutedwith halogen. In embodiments, R² is C₂-C₈ alkyl substituted with atleast one, two, three, four or five halogen. In embodiments, R² is C₂-C₈haloalkyl substituted with at least one, two, three, four or fivehalogen. In embodiments, R² is —C₂-C₈ haloalkyl. In embodiments, R² isC₂-C₆ haloalkyl. In embodiments, R² is C₂-C₄ haloalkyl. In embodiments,R² is C₂-C₄ alkyl substituted with one or more flourines. Inembodiments, R² is C₂-C₄ alkyl substituted with one, two, three, four orfive flourines. In embodiments, R² is C₂-C₄ haloalkyl substituted withone, two, three, four or five halogens. In embodiments, R² is C₂-C₄haloalkyl substituted one fluorine. In embodiments, R² is C₂-C₄haloalkyl substituted two fluorines. In embodiments, R² is C₂-C₄haloalkyl substituted three fluorines. In embodiments, R² is C₂-C₄haloalkyl substituted four fluorines. In embodiments, R² is C₂-C₄haloalkyl substituted five fluorines. In embodiments, R² is —CH₂CF₂CHF₂.In embodiments, R² is —CH(CF₃)₂.

In embodiments, R³ and R⁴ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R³ and R⁴ are joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl. Inembodiments, R³ and R⁴ are joined to form, together with the atoms towhich they are attached, a substituted or unsubstitutedheterocycloalkyl. In embodiments, R⁵ is independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl. In embodiments, R⁵ isindependently hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl. In embodiments, R³, R⁴ and R⁵are independently hydrogen, substituted or unsubstituted alkyl. Inembodiments, R³ and R⁴ are independently hydrogen, substituted orunsubstituted alkyl or substituted or unsubstituted aryl. Inembodiments, R⁵ is hydrogen or substituted or unsubstituted alkyl. Inembodiments, R³ is —C(O)R^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D),—SO₂R^(3A), —C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl. In embodiments, R⁴ is —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R⁵ is —C(O)R^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D),—SO₂R^(5A), —C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl.R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A),R^(5B), R^(5C) and R^(5D) are independently hydrogen or methyl.

In embodiments, R³, R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl. Inembodiments, R³, R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl. In embodiments, R³, R⁴ and R⁵ are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, or substituted or unsubstituted heteroaryl. In embodiments,R³, R⁴ and R⁵ are independently hydrogen, substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl. In embodiments, R³,R⁴ and R⁵ are independently hydrogen or substituted or unsubstitutedalkyl. In embodiments, R³, R⁴ and R⁵ are independently hydrogen orsubstituted or unsubstituted C₁-C₄ alkyl. In embodiments, R³, R and R⁵are independently hydrogen or unsubstituted C₁-C₄ alkyl. In embodiments,R³, R⁴ and R⁵ are independently hydrogen, methyl or ethyl. Inembodiments, R³ and R⁴ are independently substituted or unsubstitutedC₁-C₄ alkyl and R⁵ is hydrogen or substituted or unsubstituted C₁-C₄alkyl. In embodiments, R³ and R⁴ are independently hydrogen, methyl orethyl. In embodiments, R³ and R⁴ are independently methyl or ethyl. Inembodiments, R⁵ is hydrogen, methyl or ethyl. In embodiments, R³ and R⁴are independently methyl or ethyl, and R⁵ is hydrogen. In embodiments,R⁵ is hydrogen, and R³ and R⁴ are independently hydrogen, methyl orethyl. In embodiments, R⁵ is hydrogen. In embodiments, R³ and R⁴ areindependently hydrogen. In embodiments, R³, R⁴ and R⁵ are independentlyhydrogen. In embodiments, R³ and Rare independently methyl. Inembodiments, R³ and R⁴ are independently ethyl.

In embodiments, R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —N(O)_(m6), —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl. In embodiments, R⁶ is hydrogen, halogen,—CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —N(O)_(m6), —C(O)R^(6D),—C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R⁶ is hydrogen, halogen, —CX^(6.1)₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO, —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX⁶² or substituted or unsubstituted alkyl. Inembodiments, R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v1)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —NO, —NR^(6B)R^(6C), —C(O)R^(6A), —C(O)OR^(6A),—C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A), —NR^(6B)C(O)R^(6D),—NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl. In embodiments, R⁶ is hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO, —C(O)R^(6D),—C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R^(6A) and R^(6D) are independentlyhydrogen or methyl. In embodiments, R⁶ is a hydrogen, halogen, —CN,—NO₂, —COH, —COCH₃, —COOCH₃, or —COOH. In embodiments, R⁶ is halogen,—NO₂ or —COOH. In embodiments, R⁶ is halogen or —NO₂. In embodiments, R⁶is halogen or —COOH. In embodiments, R⁶ is —NO₂. In embodiments, R⁶ is ahalogen.

In embodiments, R⁷ is hydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1),—CN, —N(O)_(m7), —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX⁷²,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, or substituted or unsubstitutedheteroaryl. In embodiments, R⁷ is hydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂,—CH₂X^(7.1), —CN, —N(O)_(m7), —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1)2 or substituted or unsubstituted alkyl. In embodiments, R⁷is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —N(O)_(m7),—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R⁷ is hydrogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO, —C(O)R^(7D), —C(O)OR^(7D),—OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted or unsubstituted alkyl. Inembodiments, R^(7A) and R^(7D) are independently hydrogen or methyl. Inembodiments, R⁷ is a hydrogen, —CN, —NO₂, —COH, —COCH₃, —COOCH₃, or—COOH. In embodiments, R⁷ is a hydrogen, —NO₂ or —COOH. In embodiments,R⁷ is a hydrogen, or —NO₂. In embodiments, R⁷ is a hydrogen or —COOH. Inembodiments, R⁷ is —NO₂. In embodiments, R⁷ is a hydrogen.

In embodiments, R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1),—CN, —N(O)_(m8), —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, or substituted or unsubstitutedheteroaryl. In embodiments, R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —N(O)_(m8), —C(O)R^(8D), —C(O)ORB, —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl. In embodiments, R¹is hydrogen, —CF₃, —CCl₃, —Br₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN,—N(O)_(m8), —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ orsubstituted or unsubstituted alkyl. In embodiments, R¹ is hydrogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —NO, —C(O)R^(8D),—C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R^(8A) and R^(8D) are independentlyhydrogen or methyl. In embodiments, R⁸ is a hydrogen, —CN, —NO₂, —COH,—COCH₃, —COOCH₃, or —COOH. In embodiments, R⁸ is a hydrogen, —NO₂ or—COOH. In embodiments, R⁸ is a hydrogen, or —NO₂. In embodiments, R⁸ isa hydrogen or —COOH. In embodiments, R⁸ is —NO₂. In embodiments, R⁸ ishydrogen.

In embodiments, R⁹ is hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂,—CH₂X^(9.1), —CN, —N(O)₉, —C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl. In embodiments, R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m9), —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R⁹ is hydrogen, halogen, —CF₃,—CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —N(O), —C(O)R^(9D), —C(O)OR^(9D),—OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted or unsubstituted alkyl. Inembodiments, R⁹ is hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂,—CH₂X^(9.1), —CN, —NO, —C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂ or substituted or unsubstituted alkyl. In embodiments, R⁹is hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO,—C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R^(9A) and R^(9D) are independentlyhydrogen or methyl. In embodiments, R⁹ is a hydrogen, halogen, —CN,—NO₂, —COH, —COCH₃, —COOCH₃, or —COOH. In embodiments, R⁹ is halogen,—NO₂ or —COOH. In embodiments, R⁹ is halogen or —NO₂. In embodiments, R⁹is halogen or —COOH. In embodiments, R⁹ is —NO₂. In embodiments, R⁹ is ahalogen.

In embodiments, R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂,—CH₂X^(1.1), —CN, —N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CH₃,—(CH₂)_(n2)CX^(2.1) ₃, substituted or unsubstituted C₂-C₈ alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl; R³, R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted heterocycloalkylor substituted or unsubstituted heteroaryl; R⁶ is hydrogen, halogen,—CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —N(O)_(m6), —C(O)R^(6D),—C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl; R⁷ is hydrogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m7), —C(O)R^(7D), —C(O)OR^(7D),—OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, orsubstituted or unsubstituted heteroaryl; R¹ is hydrogen, —CX^(1.13),—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m8), —C(O)R^(8D), —C(O)OR^(8D),—OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, orsubstituted or unsubstituted heteroaryl; and R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m9), —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl.

In embodiments, R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂,—CH₂X^(1.1), —CN, —NO₂, —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂ or substituted or unsubstituted alkyl; R² is hydrogen,—CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, substituted C₂-C₆ alkyl or C₂-C₅haloalkyl; R³, R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl, wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independently hydrogen or methyl.

In embodiments, R¹ is hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂ or substituted or unsubstituted alkyl; R² is hydrogen,—CX^(2.1) ₃, —CHX^(2.1) ₂, —(CH₂)CX^(2.1) ₃, C₂-C₄ haloalkyl; R³ and R⁴are independently, unsubstituted alkyl; R⁵ is hydrogen; R⁶ is hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D),—C(O)OR^(6D), X^(6.1) ₃, —OHX^(6.1) ₂ or substituted or unsubstitutedalkyl; R⁷ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN,—NO₂, —C(O)^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ orsubstituted or unsubstituted alkyl; R¹ is hydrogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl, wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independently hydrogen or methyl, and X^(1.1), X^(2.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently are —F.

In embodiments, at least one of R¹, R⁶, R⁷, R⁸ and R⁹ are independentlyhydrogen. In embodiments, at least two of R¹, R⁶, R⁷, R⁸ and R⁹ areindependently hydrogen. In embodiments, at least three of R¹, R⁶, R⁷, R⁸and R⁹ are independently hydrogen. In embodiments, at least four of R¹,R⁶, R⁷, R⁸ and R⁹ are independently hydrogen. In embodiments, R¹, R⁶,R⁷, R⁸ and R⁹ are independently hydrogen. In embodiments, at least oneof R⁷ and R⁸ are hydrogen. In embodiments, R⁷ is hydrogen. Inembodiments, R¹ is hydrogen. In embodiments, R⁷ and R⁸ are independentlyhydrogen.

In embodiments, X is —O—; R¹ is hydrogen, halogen, —CX^(1.1) ₃,—CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂, —C(O)R^(1D), —C(O)OR^(1D),—OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted or unsubstituted alkyl; R² ishydrogen, —CX^(2.1) ₃, —CH₃, —(CH₂)_(n2)CX^(2.1) ₃, substituted orunsubstituted C₂-C₆ alkyl, substituted or unsubstituted aryl or C₂-C₆haloalkyl; R³ and R⁴ are independently substituted or unsubstitutedalkyl or substituted or unsubstituted aryl; R⁵ is hydrogen orsubstituted or unsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1)₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ ishydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂,—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X—^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R^(1D), R^(6D), R^(7D), R^(8D) andR^(9D) are independent hydrogen or methyl.

In embodiments, X is —O—; R¹ is hydrogen, halogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(1D), —C(O)OR^(1D), —OCF₃, —OCHF₂,—OCCl₃, —OCBr₃ or substituted or unsubstituted alkyl; R² is hydrogen,—CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, substituted C₂-C₆ alkyl, substitutedor unsubstituted aryl or haloalkyl; R³ and R⁴ are independentlyhydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aryl; R⁵ is hydrogen or substituted or unsubstitutedalkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1),—CN, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ orsubstituted or unsubstituted alkyl; R⁷ is hydrogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂ or substituted or unsubstituted alkyl; R¹ is hydrogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(8D),—C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl; and R⁹ is hydrogen, halogen, —CF₃, —CCl₃, —CCl₃,—CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂ or substituted or unsubstituted alkyl. In embodiments,R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D) are independent hydrogen ormethyl.

In embodiments, at least two of R¹, R⁶, R⁷, R⁸ and R⁹ are independentlyhydrogen. In embodiments, two of R¹, R⁶, R⁷, R⁸ and R⁹ are independentlyhydrogen. In embodiments, R⁶, R⁷, R⁸ and R⁹ are independently hydrogenand R¹ is —NO₂. In embodiments, R⁶, R⁷, R⁸ and R⁹ are independentlyhydrogen and R¹ is —NO₂. In embodiments, R⁶, R⁷, R⁸ and R⁹ areindependently hydrogen and R¹ is —COOH. In embodiments, R⁶, R⁷, R⁸ andR⁹ are independently hydrogen and R¹ is —F. In embodiments, R¹, R⁷, R⁸and R⁹ are independently hydrogen and R⁶ is —COOH. In embodiments, R¹,R⁷, R⁸ and R⁹ are independently hydrogen and R⁶ is —F. In embodiments,R¹, R⁷, R⁸ and R⁹ are independently hydrogen and R⁶ is —Cl. Inembodiments, R¹, R⁷, R⁸ and R⁹ are independently hydrogen and R⁶ is—NO₂, with proviso that when X is —O—; R² is —CH₂CF₃; one of R³ and R ishydrogen and one of R³ and R⁴ is unsubstituted C₁-C₃ alkyl, then R⁶ isnot —NO₂. In embodiments, R¹, R⁶, R⁷ and R⁸ are independently hydrogenand R⁹ is —COOH. In embodiments, R¹, R⁶, R⁷ and R⁸ are independentlyhydrogen and R⁹ is —F. In embodiments, R¹, R⁶, R⁷ and R⁸ areindependently hydrogen and R⁹ is —Cl. In embodiments, R¹, R⁶, R⁷ and R⁸are independently hydrogen and R⁹ is —NO₂, with proviso that when X is—O—; R² is —CH₂CF₃; one of R³ and R is hydrogen and one of R³ and R⁴ isunsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂. In embodiments, R¹, R⁶,R⁷, R⁸ and R⁹ are independently hydrogen. In embodiments, R¹ is nothydrogen and R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

In embodiments, the compound has Formula IA:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are as described herein.

In embodiments, R² is —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, or C₂-C₈haloalkyl. In embodiments, R³, R⁴ and R⁵ are independently hydrogen,substituted or unsubstituted alkyl. In embodiments, R¹ is hydrogen,halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂, —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), X^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(1D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R⁷ and R⁸ are independentlyhydrogen. In embodiments, R³ and R⁴ substituted or unsubstituted alkyl;R⁵ are independently hydrogen; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX⁶² or substituted or unsubstituted alkyl; R⁷ is hydrogen, —CX^(7.1)₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D), —C(O)OR^(7D),—OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted or unsubstituted alkyl; R⁸ ishydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —NO₂,—C(O)R^(8D), —C(O)OR^(8D), —OCX^(1.13), —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl; and R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂ or substituted or unsubstituted alkyl; wherein R^(1D),R^(6D), R^(7D), R^(8D) and R^(9D) are independently hydrogen or methyl,and X^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1), and X^(9.1) areindependently are —F. In embodiments, R⁷ and R⁸ are independentlyhydrogen. In embodiments, R³ and R⁴ are independently methyl or ethyl.In embodiment, R⁵ is hydrogen. In embodiments, R¹ is hydrogen, halogen,—NO₂, or —⁻COOH; R⁵ is hydrogen; R⁶ is hydrogen, halogen —NO₂, or —COOH;R⁷ and R⁸ are independently hydrogen; and R⁹ is hydrogen, halogen NO₂,or —COOH. In embodiments, R¹, R⁷ and R⁸ are independently hydrogen. Inembodiments, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen. Inembodiments, R¹, R⁷, R⁸ and R⁹ are independently hydrogen. Inembodiments, R¹, R⁶, R⁷ and R⁸ are independently hydrogen. Inembodiments, R¹, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

In embodiments, R³ and R⁴ are independently hydrogen, methyl or ethyl.In embodiments, R² is C₂-C₄ alkyl substituted with fluorines. Inembodiments, R² is C₂-C₄ alkyl substituted with at least one, two,three, four or five fluorines. In embodiments, R² is C₂-C₄ haloalkylsubstituted with at least one, two, three, four or five fluorines. Inembodiments, R¹, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen. Inembodiments, R¹ and R⁶ are joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted pyrazolyl,oxazolyl, or thiazolyl; R² is C₂-C₄ haloalkyl; R³ and R⁴ areindependently hydrogen, methyl or ethyl. The compound of claim 16,wherein the compound is represented with Formula IB, IC, ID, or IE:

In embodiments, R⁵, R⁷, R⁸ and R⁹ are independently hydrogen. Inembodiments, R² is —CH(CF₃)₂ or —CH₂(CF₂)₂H.

In embodiments, R² is —CX^(2.1) ₃, or C₂-C₄ haloalkyl. In embodiments,R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(6.1), —CN,—NO₂, —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R³, R⁴ and R⁵ are independentlyhydrogen, substituted or unsubstituted alkyl, or R³ and R⁴ areoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO₂,—C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted orunsubstituted alkyl; R⁷ is hydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂,—CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂ or substituted or unsubstituted alkyl; R¹ is hydrogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D),—C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl; and R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D), —C(O)OR^(9D),—OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted or unsubstituted alkyl. Inembodiments, R⁷ and R⁸ are independently hydrogen. In embodiments, R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂,—C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R³ and R⁴ substituted or unsubstituted alkyl; R⁵are independently hydrogen; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ ishydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂,—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl; wherein RID, R^(6D), R^(7D), RD and R^(9D) areindependently hydrogen or methyl, and X^(1.1), X^(2.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently are —F. In embodiments,R³ and R⁴ are independently hydrogen, methyl or ethyl. In embodiments,R¹ is hydrogen, halogen, —NO₂, —COOH; R⁵ is hydrogen; R⁶ is hydrogen,halogen —NO₂, or —COOH; R⁷ is hydrogen; R¹ is hydrogen; and R⁹ ishydrogen, halogen NO₂, or —COOH. In embodiments, R¹ is a hydrogen,halogen, COOH or —NO₂; R² is C₂-C₄ haloalkyl; R⁵ is hydrogen; R⁶ is ahydrogen, halogen, COOH or —NO₂; R⁷ and R⁸ are independently hydrogen;R⁹ is a hydrogen, halogen, COOH or —NO₂. In embodiments, R³ and R⁴ areindependently hydrogen, methyl or ethyl. In embodiments, R² is alkylsubstituted with at least one fluorine. In embodiments, R¹, R⁶, R⁷, R⁸and R⁹ are independently hydrogen.

In embodiments, R¹ is independently hydrogen, halogen, —CX^(1.1) ₃,—CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C),—NHNR^(1B)R^(1C), —ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C),—NHC(O)NR^(1B)R^(1C), —N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D),—C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),R^(1E)-substituted or unsubstituted alkyl, R^(1E)-substituted orunsubstituted heteroalkyl, R^(1E)-substituted or unsubstitutedcycloalkyl, R^(1E)-substituted or unsubstituted heterocycloalkyl,R^(1E)-substituted or unsubstituted aryl, or R^(1E)-substituted orunsubstituted heteroaryl. In embodiments, R¹ is independently hydrogen,halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A),—SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C), —ONR^(1B)R^(1C),—NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C), —N(O)_(m1),—NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A),—NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D),—NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ (e.g. hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂), R^(1E)-substituted or unsubstituted C₁-C₆alkyl, R^(1E)-substituted or unsubstituted 2 to 6 membered heteroalkyl,R^(1E)-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(1E)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(1E)-substituted orunsubstituted phenyl, or R^(1E)-substituted or unsubstituted 5 to 6membered heteroaryl.

R^(1E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(1F)-substituted or unsubstituted alkyl, R^(1F)-substituted orunsubstituted heteroalkyl, R^(1F)-substituted or unsubstitutedcycloalkyl, R^(1F)-substituted or unsubstituted heterocycloalkyl,R^(1F)-substituted or unsubstituted aryl, or R^(1F)-substituted orunsubstituted heteroaryl. In embodiments, R^(1E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(1F)-substituted orunsubstituted C₁-C₆ alkyl, R^(1F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(1F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(1F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(1F)-substituted or unsubstituted phenyl, orR^(1F-)substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R² is independently hydrogen, halogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —CH₂X^(2.1), —CN, —SO_(n2)R^(2A), —SO_(v2)NR^(2B)R^(2C),—NHNR^(2B)R^(2C), —ONR^(2B)R^(2C), —NHC(O)NHNR^(2B)R^(2C),—NHC(O)NR^(2B)R^(2C), —N(O)_(m2), —NR^(2B)R^(2C), —C(O)R^(2D),—C(O)OR^(2D), —C(O)NR^(2B)R^(2C), —OR^(2A), —NR^(2B)SO₂R^(2A),—NR^(2B)C(O)R^(2D), —NR^(2B)C(O)OR^(2D), —NR^(2B)OR^(2D), —OCX^(2.1) ₃,—OCHX^(2.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂), —OCHI₂,R^(2E)-substituted or unsubstituted alkyl, R^(2E)-substituted orunsubstituted heteroalkyl, R^(2E)-substituted or unsubstitutedcycloalkyl, R^(2E)-substituted or unsubstituted heterocycloalkyl,R^(2E)-substituted or unsubstituted aryl, or R^(2E)-substituted orunsubstituted heteroaryl. In embodiments, R² is independently hydrogen,halogen, —CX^(2.1) ₃, —CHX^(2.1) ₂, —CH₂X^(2.1), —CN, —SO_(n2)R^(2A),—SO_(v2)NR^(2B)R^(2C), —NHNR^(2B)R^(2C), —ONR^(2B)R^(2C),—NHC(O)NHNR^(2B)R^(2C), —NHC(O)NR^(2B)R^(2C), —N(O)_(m2),—NR^(2B)R^(2C), —C(O)R^(2D), —C(O)OR^(2D), —C(O)NR^(2B)R^(2C), —OR^(2A),—NR^(2B)SO₂R^(2A), —NR^(2B)C(O)R^(2D), —NR^(2B)C(O)OR^(2D),—NR^(2B)OR^(2D), —OCX^(2.1) ₃, —OCHX^(2.1) ₂ (e.g. hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂), R^(2E)-substituted or unsubstituted C₁-C₆alkyl, R^(2E)-substituted or unsubstituted 2 to 6 membered heteroalkyl,R^(2E)-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(2E)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(2E)-substituted orunsubstituted phenyl, or R^(2E)-substituted or unsubstituted 5 to 6membered heteroaryl. In embodiments, R² is haloalkyl. In embodiments, R²is C₂-C₈ haloalkyl. In embodiments, R² is C₂-C₆ haloalkyl. Inembodiments, R² is C₂-C₄ haloalkyl. In embodiments, R² is C₂-C₄haloalkyl including at least one, two, three, four or five fluorines.

R^(2E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(2F)-substituted or unsubstituted alkyl, R^(2F)-substituted orunsubstituted heteroalkyl, R^(2F)-substituted or unsubstitutedcycloalkyl, R^(2F)-substituted or unsubstituted heterocycloalkyl,R^(2F)-substituted or unsubstituted aryl, or R^(2F)-substituted orunsubstituted heteroaryl. In embodiments, R^(2E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(2F)-substituted orunsubstituted C₁-C₆ alkyl, R^(2F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(2F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(2F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(2F)-substituted or unsubstituted phenyl, orR^(2F)-substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R³ is independently hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),R^(3E)-substituted or unsubstituted alkyl, R^(3E)-substituted orunsubstituted heteroalkyl, R^(3E)-substituted or unsubstitutedcycloalkyl, R^(3E)-substituted or unsubstituted heterocycloalkyl,R^(3E)-substituted or unsubstituted aryl, or R^(3E)-substituted orunsubstituted heteroaryl. In embodiments, R³ is independently hydrogen,R^(3E)-substituted or unsubstituted C₁-C₆ alkyl, R^(3E)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(3E)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(3E)-substituted or unsubstituted 3 to6 membered heterocycloalkyl, R^(3E)-substituted or unsubstituted phenyl,or R^(3E)-substituted or unsubstituted 5 to 6 membered heteroaryl.

R^(3E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(3F)-substituted or unsubstituted alkyl, R^(3F)-substituted orunsubstituted heteroalkyl, R^(3F)-substituted or unsubstitutedcycloalkyl, R^(3F)-substituted or unsubstituted heterocycloalkyl,R^(3F)-substituted or unsubstituted aryl, or R^(3F)-substituted orunsubstituted heteroaryl. In embodiments, R^(3E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂), R^(3F)-substituted orunsubstituted C₁-C₆ alkyl, R^(3F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(3F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(3F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(3F)-substituted or unsubstituted phenyl, orR^(3F)-substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁴ is independently hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(3C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),R^(4E)-substituted or unsubstituted alkyl, R^(4E)-substituted orunsubstituted heteroalkyl, R^(4E)-substituted or unsubstitutedcycloalkyl, R^(4E)-substituted or unsubstituted heterocycloalkyl,R^(4E)-substituted or unsubstituted aryl, or R^(4E)-substituted orunsubstituted heteroaryl. In embodiments, R¹ is independently hydrogen,R^(4E)-substituted or unsubstituted C₁-C₆ alkyl, R^(4E)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(4E)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(4E)-substituted or unsubstituted 3 to6 membered heterocycloalkyl, R^(4E)-substituted or unsubstituted phenyl,or R^(4E)-substituted or unsubstituted 5 to 6 membered heteroaryl.

R^(4E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(4F)-substituted or unsubstituted alkyl, R^(4F)-substituted orunsubstituted heteroalkyl, R^(4F)-substituted or unsubstitutedcycloalkyl, R^(4F)-substituted or unsubstituted heterocycloalkyl,R^(4F)-substituted or unsubstituted aryl, or R^(4F)-substituted orunsubstituted heteroaryl. In embodiments, R^(4E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(4F)-substituted orunsubstituted C₁-C₆ alkyl, R^(4F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(4F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(4F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(4F)-substituted or unsubstituted phenyl, orR^(4F-)substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R³ and R⁴ are joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted (e.g. C₃-C₆)cycloalkyl, a substituted or unsubstituted (e.g. 3 to 6 membered)heterocycloalkyl, substituted or unsubstituted (e.g. phenyl) aryl orsubstituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl. Inembodiments, R³ and R⁴ are joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted (e.g. C₃-C₆)cycloalkyl, a substituted or unsubstituted (e.g. 3 to 6 membered)heterocycloalkyl or substituted or unsubstituted (e.g. 5 to 6 membered)heteroaryl. In embodiments, R³ and R⁴ are joined to form, together withthe atoms to which they are attached, a substituted or unsubstituted(e.g. C₃-C₆) cycloalkyl or a substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl. In embodiments, R³ and R⁴ are joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted (e.g. 3 to 6 membered) heterocycloalkyl. Inembodiments, R³ and R⁴ are joined to form, together with the atoms towhich they are attached, R^(3E)-substituted or unsubstituted (e.g.C₃-C₆) cycloalkyl, R^(3E)-substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl, R^(3E)-substituted or unsubstituted (e.g.phenyl) aryl or R^(3E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R³ and R⁴ are joined to form,together with the atoms to which they are attached, R^(3E)-substitutedor unsubstituted (e.g. C₃-C₆) cycloalkyl, R^(3E)-substituted orunsubstituted (e.g. 3 to 6 membered) heterocycloalkyl orR^(3E)-substituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl.In embodiments, R³ and R⁴ are joined to form, together with the atoms towhich they are attached, R^(3E)-substituted or unsubstituted (e.g.C₃-C₆) cycloalkyl or R^(3E)-substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl. In embodiments, R³ and R⁴ are joined toform, together with the atoms to which they are attached,R^(3E)-substituted or unsubstituted (e.g. 3 to 6 membered)heterocycloalkyl. In embodiments, R³ and R⁴ are joined to form, togetherwith the atoms to which they are attached, R^(3E)-substituted orunsubstituted piperidinyl or morpholinyl. In embodiments, R³ and R⁴ arejoined to form, together with the atoms to which they are attached,unsubstituted piperidinyl or morpholinyl. In embodiments, the compoundis

In embodiments, R⁵ is independently hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR, —SO₂R^(5A), —C(O)NR^(8B)R^(8C),R^(5E)-substituted or unsubstituted alkyl, R^(5E)-substituted orunsubstituted heteroalkyl, R^(5E)-substituted or unsubstitutedcycloalkyl, R^(5E)-substituted or unsubstituted heterocycloalkyl,R^(5E)-substituted or unsubstituted aryl, or R^(5E)-substituted orunsubstituted heteroaryl. In embodiments, R⁵ is independently hydrogen,RE-substituted or unsubstituted C₁-C₆ alkyl, R^(5E)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(5E) substituted orunsubstituted C₃-C₆ cycloalkyl, R^(5E)-substituted or unsubstituted 3 to6 membered heterocycloalkyl, R^(5E)-substituted or unsubstituted phenyl,or R^(5E)-substituted or unsubstituted 5 to 6 membered heteroaryl.

R^(5E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(5F)-substituted or unsubstituted alkyl, R⁵-substituted orunsubstituted heteroalkyl, R⁵-substituted or unsubstituted cycloalkyl,R^(5F)-substituted or unsubstituted heterocycloalkyl, R⁵-substituted orunsubstituted aryl, or R^(5F)-substituted or unsubstituted heteroaryl.In embodiments, R is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R⁵-substituted or unsubstituted C₁-C₆ alkyl, R⁵-substituted orunsubstituted 2 to 6 membered heteroalkyl, R⁵-substituted orunsubstituted C₃-C₆ cycloalkyl, R⁵-substituted or unsubstituted 3 to 6membered heterocycloalkyl, R^(5F)-substituted or unsubstituted phenyl,or R^(5F)-substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁶ is independently hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),R^(6E)-substituted or unsubstituted alkyl, R^(6E)-substituted orunsubstituted heteroalkyl, R^(6E)-substituted or unsubstitutedcycloalkyl, R^(6E)-substituted or unsubstituted heterocycloalkyl,R^(6E)-substituted or unsubstituted aryl, or R^(6E)-substituted orunsubstituted heteroaryl. In embodiments, R⁶ is independently hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A),—SO_(v6)NR^(6B)R^(6C), —NHNR^(6B)R^(6C), —ONR^(6B)R^(6C),—NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C), —N(O)_(m6),—NR^(6B)R^(6C), —C(O)R^(6D), —C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A),—NR^(6B)SO₂R^(6A), —NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D),—NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ (e.g. hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂), R^(6E)-substituted or unsubstituted C₁-C₆alkyl, R^(6E)-substituted or unsubstituted 2 to 6 membered heteroalkyl,R^(6E)-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(6E)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(6E)-substituted orunsubstituted phenyl, or R^(6E)-substituted or unsubstituted 5 to 6membered heteroaryl.

R^(6E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(6F)-substituted or unsubstituted alkyl, R^(6F)-substituted orunsubstituted heteroalkyl, R^(6F)-substituted or unsubstitutedcycloalkyl, R^(6F)-substituted or unsubstituted heterocycloalkyl,R^(6F)-substituted or unsubstituted aryl, or R^(6F)-substituted orunsubstituted heteroaryl. In embodiments, R^(6E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(6F)-substituted orunsubstituted C₁-C₆ alkyl, R^(6F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(6F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(6F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(6F)-substituted or unsubstituted phenyl, orR^(6F)-substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁷ is independently hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7B)C(O)R^(7D), —NR^(7B)C(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),R^(7E)-substituted or unsubstituted alkyl, R^(7E)-substituted orunsubstituted heteroalkyl, R^(7E)-substituted or unsubstitutedcycloalkyl, R^(7E)-substituted or unsubstituted heterocycloalkyl,R^(7E)-substituted or unsubstituted aryl, or R^(7E)-substituted orunsubstituted heteroaryl. In embodiments, R⁷ is independently hydrogen,halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n7)R^(7A),—SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C), —ONR^(7B)R^(7C),—NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C), —N(O)_(m7),—NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A),—NR^(7B)SO₂R^(7A), —NR^(7B)C(O)R^(7D), —NR^(7B)C(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ (e.g. hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂), R^(7E)-substituted or unsubstituted C₁-C₆alkyl, R^(7E)-substituted or unsubstituted 2 to 6 membered heteroalkyl,R^(7E)-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(7E)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(7E)-substituted orunsubstituted phenyl, or R^(7E)-substituted or unsubstituted 5 to 6membered heteroaryl.

R^(7E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(7F)-substituted or unsubstituted alkyl, R^(7F)-substituted orunsubstituted heteroalkyl, R^(7F)-substituted or unsubstitutedcycloalkyl, R^(7F)-substituted or unsubstituted heterocycloalkyl,R^(7F)-substituted or unsubstituted aryl, or R^(7F)-substituted orunsubstituted heteroaryl. In embodiments, R^(7E) is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(7F)-substituted orunsubstituted C₁-C₆ alkyl, R^(7F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(7F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(7F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(7F)-substituted or unsubstituted phenyl, orR^(7F)-substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁸ is independently hydrogen, halogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(1B)R^(1C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(1B)R^(1C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),R^(8E)-substituted or unsubstituted alkyl, R^(8E)-substituted orunsubstituted heteroalkyl, R^(8E)-substituted or unsubstitutedcycloalkyl, R^(8E)-substituted or unsubstituted heterocycloalkyl,R^(8E)-substituted or unsubstituted aryl, or R^(8E)-substituted orunsubstituted heteroaryl. In embodiments, R¹ is independently hydrogen,halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A),—SO_(v8)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m8),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(1D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ (e.g. hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂), R^(8E)-substituted or unsubstituted C₁-C₆alkyl, R^(8E)-substituted or unsubstituted 2 to 6 membered heteroalkyl,R^(8E)-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(8E)-substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, R^(8E)-substituted orunsubstituted phenyl, or R^(8E)-substituted or unsubstituted 5 to 6membered heteroaryl.

R^(8E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(8F)-substituted or unsubstituted alkyl, RF-substituted orunsubstituted heteroalkyl, RF-substituted or unsubstituted cycloalkyl,RF-substituted or unsubstituted heterocycloalkyl, RF-substituted orunsubstituted aryl, or RF-substituted or unsubstituted heteroaryl. Inembodiments, R^(8E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃,—Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R⁸-substituted or unsubstituted C₁-C₆ alkyl,R-substituted or unsubstituted 2 to 6 membered heteroalkyl,RF-substituted or unsubstituted C₃-C₆ cycloalkyl, R^(8F)-substituted orunsubstituted 3 to 6 membered heterocycloalkyl, R^(8F)-substituted orunsubstituted phenyl, or R^(8F)-substituted or unsubstituted 5 to 6membered heteroaryl.

In embodiments, R⁹ is independently hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),RE-substituted or unsubstituted alkyl, RE-substituted or unsubstitutedheteroalkyl, RE-substituted or unsubstituted cycloalkyl, RE-substitutedor unsubstituted heterocycloalkyl, R^(9E)-substituted or unsubstitutedaryl, or R^(9E)-substituted or unsubstituted heteroaryl. In embodiments,R⁹ is independently hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂,—CH₂X^(9.1), —CN, —SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂ (e.g. hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂),RE-substituted or unsubstituted C₁-C₆ alkyl, RE-substituted orunsubstituted 2 to 6 membered heteroalkyl, RE-substituted orunsubstituted C₃-C₆ cycloalkyl, RE-substituted or unsubstituted 3 to 6membered heterocycloalkyl, RE-substituted or unsubstituted phenyl, orRE-substituted or unsubstituted 5 to 6 membered heteroaryl.

R^(9E) is independently oxo, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(9F)-substituted or unsubstituted alkyl, R^(9F)-substituted orunsubstituted heteroalkyl, R^(9F)-substituted or unsubstitutedcycloalkyl, R^(9F)-substituted or unsubstituted heterocycloalkyl,R^(9F)-substituted or unsubstituted aryl, or R^(9F)-substituted orunsubstituted heteroaryl. In embodiments, R⁹ is independently oxo,halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, R^(9F)-substituted orunsubstituted C₁-C₆ alkyl, R^(9F)-substituted or unsubstituted 2 to 6membered heteroalkyl, R^(9F)-substituted or unsubstituted C₃-C₆cycloalkyl, R^(9F)-substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, R^(9F)-substituted or unsubstituted phenyl, orR^(9F-)substituted or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R^(1A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(1AF)-substituted or unsubstituted alkyl,R^(1AF)-substituted or unsubstituted heteroalkyl, R^(1AF)-substituted orunsubstituted cycloalkyl, R^(1AF)-substituted or unsubstitutedheterocycloalkyl, R^(1AF)-substituted or unsubstituted aryl, orR^(1AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(1AF)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(1AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(1AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(1AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(1AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(1AF)-substituted or unsubstitutedphenyl, or R^(1AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(1B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(1BF)-substituted or unsubstituted alkyl,R^(1BF)-substituted or unsubstituted heteroalkyl, R^(1BF)-substituted orunsubstituted cycloalkyl, R^(1BF)-substituted or unsubstitutedheterocycloalkyl, R^(1BF)-substituted or unsubstituted aryl, orR^(1BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(1B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(1BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(1BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(1BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(1BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(1BF)-substituted or unsubstitutedphenyl, or R^(1BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(1C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(1CF)-substituted or unsubstituted alkyl,R^(1CF)-substituted or unsubstituted heteroalkyl, R^(1CF)-substituted orunsubstituted cycloalkyl, R^(1CF)-substituted or unsubstitutedheterocycloalkyl, R^(1AF)-substituted or unsubstituted aryl, orR^(1CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(1C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(1CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(1CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(1CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(1AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(1CF)-substituted or unsubstitutedphenyl, or R^(1CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(1B) and R^(1C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(1AF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(1C-)substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(1D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(1DF)-substituted or unsubstituted alkyl,R^(1DF)-substituted or unsubstituted heteroalkyl, R^(1DF)-substituted orunsubstituted cycloalkyl, R^(1DF)-substituted or unsubstitutedheterocycloalkyl, R^(1DF)—Substituted or unsubstituted aryl, orR^(1DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(1D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(1DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(1DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(1DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(1DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(1DF)-substituted or unsubstitutedphenyl, or R^(1DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(2A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(2AF)-substituted or unsubstituted alkyl,R^(2AF)-substituted or unsubstituted heteroalkyl, R^(2AF)-substituted orunsubstituted cycloalkyl, R^(2AF)-substituted or unsubstitutedheterocycloalkyl, R^(2AF)-substituted or unsubstituted aryl, orR^(2AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(2A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(2AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(2AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(2AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(2AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(2AF)-substituted or unsubstitutedphenyl, or R^(2AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(2B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(2BF)-substituted or unsubstituted alkyl,R^(2BF)-substituted or unsubstituted heteroalkyl, R^(2BF)-substituted orunsubstituted cycloalkyl, R^(2BF)-substituted or unsubstitutedheterocycloalkyl, R^(2BF)-substituted or unsubstituted aryl, orR^(2F)-substituted or unsubstituted heteroaryl. In embodiments, R^(2B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(2BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(2BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(2BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(2BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(2BF)-substituted or unsubstitutedphenyl, or R^(2BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(2C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(2CF)-substituted or unsubstituted alkyl,R^(2CF)-substituted or unsubstituted heteroalkyl, R^(2CF)-substituted orunsubstituted cycloalkyl, R^(2CF)-substituted or unsubstitutedheterocycloalkyl, R^(2CF)-substituted or unsubstituted aryl, orR^(2CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(2C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(2CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(2CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(2CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(2CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(2CF)-substituted or unsubstitutedphenyl, or R^(2CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(2B) and R^(2C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(2CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(2CF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(2D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(2DF)-substituted or unsubstituted alkyl,R^(2DF)-substituted or unsubstituted heteroalkyl, R^(2DF)-substituted orunsubstituted cycloalkyl, R^(2DF)-substituted or unsubstitutedheterocycloalkyl, R^(2DF)-substituted or unsubstituted aryl, orR^(2DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(2DF)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —Br₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(2DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(2DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(2DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(2DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(2DF)-substituted or unsubstitutedphenyl, or R^(2DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(3A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(3AF)-substituted or unsubstituted alkyl,R^(3AF)-substituted or unsubstituted heteroalkyl, R^(3AF)-substituted orunsubstituted cycloalkyl, R^(3AF)-substituted or unsubstitutedheterocycloalkyl, R^(3AF)-substituted or unsubstituted aryl, orR^(3AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(3A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(3AF)-substituted or unsubstituted C₁-C₆ alkyl, R³-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(3AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(3AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(3AF)-substituted or unsubstitutedphenyl, or R^(3AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(3B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(3BF)-substituted or unsubstituted alkyl,R^(3BF)-substituted or unsubstituted heteroalkyl, R^(3BF)-substituted orunsubstituted cycloalkyl, R^(3BF)-substituted or unsubstitutedheterocycloalkyl, R^(3BF)-substituted or unsubstituted aryl, orR^(3BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(3B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(3BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(3BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R³⁻substituted orunsubstituted C₃-C₆ cycloalkyl, R^(3BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(3BF)-substituted or unsubstitutedphenyl, or R^(3BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(3C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R³CF-substituted or unsubstituted alkyl,R^(3CF)-substituted or unsubstituted heteroalkyl, R^(3CF)-substituted orunsubstituted cycloalkyl, R^(3CF)-substituted or unsubstitutedheterocycloalkyl, R^(3CF)-substituted or unsubstituted aryl, orR^(3CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(3C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R³CF-substituted or unsubstituted C₁-C₆ alkyl, R^(3CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(3C)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(3CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(3CF)-substituted or unsubstitutedphenyl, or R^(3CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(3B) and R^(3C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(3CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(3C)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(3D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(3DF)-substituted or unsubstituted alkyl,R^(3DF)-substituted or unsubstituted heteroalkyl, R^(3DF)-substituted orunsubstituted cycloalkyl, R^(3DF)-substituted or unsubstitutedheterocycloalkyl, R^(3DF)-substituted or unsubstituted aryl, orR^(3DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(3D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(3DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(3DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(3DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(3D)-substituted or unsubstituted 3 to6 membered heterocycloalkyl, R^(3DF)-substituted or unsubstitutedphenyl, or R^(3DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(4A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(4AF)-substituted or unsubstituted alkyl,R^(4AF)-substituted or unsubstituted heteroalkyl, R^(4AF)-substituted orunsubstituted cycloalkyl, R^(4AF)-substituted or unsubstitutedheterocycloalkyl, R^(4AF)-substituted or unsubstituted aryl, orR^(4AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(4A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(4AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(4AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(4AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(4AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(4AF)-substituted or unsubstitutedphenyl, or R^(4AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(4B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(4BF)-substituted or unsubstituted alkyl,R^(4BF)-substituted or unsubstituted heteroalkyl, R^(4BF)-substituted orunsubstituted cycloalkyl, R^(4BF)-substituted or unsubstitutedheterocycloalkyl, R^(4BF)-substituted or unsubstituted aryl, orR^(4BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(4B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R⁴BF-substituted or unsubstituted C₁-C₆ alkyl, R^(4BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(4BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(4BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(4BF)-substituted or unsubstitutedphenyl, or R^(4BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(4C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(4CF)-substituted or unsubstituted alkyl,R^(4CF)-substituted or unsubstituted heteroalkyl, R^(4CF)-substituted orunsubstituted cycloalkyl, R^(4CF)-substituted or unsubstitutedheterocycloalkyl, R^(4CF)-substituted or unsubstituted aryl, orR^(4CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(4C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(4CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(4CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(4CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(4CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(4CF)-substituted or unsubstitutedphenyl, or R^(4CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(4B) and R^(4C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(4CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(4CF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(4D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(4DF)-substituted or unsubstituted alkyl,R^(4DF)-substituted or unsubstituted heteroalkyl, R^(4DF)-substituted orunsubstituted cycloalkyl, R^(4DF)-substituted or unsubstitutedheterocycloalkyl, R^(4DF)-substituted or unsubstituted aryl, orR^(4DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(4D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(4DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(4DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(4DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(4DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(4DF)-substituted or unsubstitutedphenyl, or R^(4DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(5A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(5AF)-substituted or unsubstituted alkyl,R^(5AF)-substituted or unsubstituted heteroalkyl, R^(5AF)-substituted orunsubstituted cycloalkyl, R^(5AF)-substituted or unsubstitutedheterocycloalkyl, R^(5AF)-substituted or unsubstituted aryl, orR^(5AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(5A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(5AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(5AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(5AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(5AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(5AF)-substituted or unsubstitutedphenyl, or R^(5AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(5B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(5BF)-substituted or unsubstituted alkyl,R^(5BF)-substituted or unsubstituted heteroalkyl, R^(5BF)-substituted orunsubstituted cycloalkyl, R^(5BF)-substituted or unsubstitutedheterocycloalkyl, R^(5BF)-substituted or unsubstituted aryl, orR^(5BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(5B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(5BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(5BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(5BF) substituted orunsubstituted C₃-C₆ cycloalkyl, R^(5BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(5BF)-substituted or unsubstitutedphenyl, or R^(5BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(5C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(5CF)-substituted or unsubstituted alkyl,R^(5CF)-substituted or unsubstituted heteroalkyl, R^(5CF)-substituted orunsubstituted cycloalkyl, R^(5CF)-substituted or unsubstitutedheterocycloalkyl, R^(5CF)-substituted or unsubstituted aryl, orR^(5CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(5CF)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(5CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(5CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(5CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(5CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(5CF)-substituted or unsubstitutedphenyl, or R^(5CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(5B) and R^(5C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(5CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(5CF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(5D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(5DF)-substituted or unsubstituted alkyl,R^(5DF)-substituted or unsubstituted heteroalkyl, R^(5DF)-substituted orunsubstituted cycloalkyl, R^(5DF)-substituted or unsubstitutedheterocycloalkyl, R^(5DF)-substituted or unsubstituted aryl, orR^(5DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(5D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(5DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(5DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(5DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(5DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(5DF)-substituted or unsubstitutedphenyl, or R^(5DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(6A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(6AF)-substituted or unsubstituted alkyl,R^(6AF)-substituted or unsubstituted heteroalkyl, R^(6AF)-substituted orunsubstituted cycloalkyl, R^(6AF)-substituted or unsubstitutedheterocycloalkyl, R^(6AF)-substituted or unsubstituted aryl, orR^(6AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(6A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(6AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(6AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(6AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(6AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(6AF)-substituted or unsubstitutedphenyl, or R^(6AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(6B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(6B)F-substituted or unsubstituted alkyl,R^(6BF)-substituted or unsubstituted heteroalkyl, R^(6BF)-substituted orunsubstituted cycloalkyl, R^(6BF)-substituted or unsubstitutedheterocycloalkyl, R^(6BF)-substituted or unsubstituted aryl, orR^(6BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(6B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(6B)F-substituted or unsubstituted C₁-C₆ alkyl, R^(6BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(6BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(6BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(6BF)-substituted or unsubstitutedphenyl, or R^(6BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(6C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(6CF)-substituted or unsubstituted alkyl,R^(6CF)-substituted or unsubstituted heteroalkyl, R^(6C)-substituted orunsubstituted cycloalkyl, R^(6CF)-substituted or unsubstitutedheterocycloalkyl, R^(6CF)-substituted or unsubstituted aryl, orR^(6CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(6C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(6CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(6BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(6C)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(6BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(6CF)-substituted or unsubstitutedphenyl, or R^(6CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(6B) and R^(6C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(6BF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(6BF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(6D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(6DF)-substituted or unsubstituted alkyl,R^(6DF)-substituted or unsubstituted heteroalkyl, R^(6DF)-substituted orunsubstituted cycloalkyl, R^(6DF)-substituted or unsubstitutedheterocycloalkyl, R^(6DF)-substituted or unsubstituted aryl, orR^(6DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(6D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(6DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(6DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(6DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(6DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(6DF)-substituted or unsubstitutedphenyl, or R^(6DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(7A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(7AF)-substituted or unsubstituted alkyl,R^(7AF)-substituted or unsubstituted heteroalkyl, R^(7AF)-substituted orunsubstituted cycloalkyl, R^(7AF)-substituted or unsubstitutedheterocycloalkyl, R^(7AF)-substituted or unsubstituted aryl, orR^(7AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(7A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(7F)-substituted or unsubstituted C₁-C₆ alkyl, R^(7AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(7AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(7AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(7AF)-substituted or unsubstitutedphenyl, or R^(7AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(7B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(7AF)-substituted or unsubstituted alkyl,R^(7BF)-substituted or unsubstituted heteroalkyl, R^(7BF)-substituted orunsubstituted cycloalkyl, R^(7BF)-substituted or unsubstitutedheterocycloalkyl, R^(7BF)-substituted or unsubstituted aryl, orR^(7BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(7B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(7BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(7BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R⁷BF substituted orunsubstituted C₃-C₆ cycloalkyl, R^(7BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(7BF)-substituted or unsubstitutedphenyl, or R^(7BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(7C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(7CF)-substituted or unsubstituted alkyl,R^(7CF)-substituted or unsubstituted heteroalkyl, R^(7CF)-substituted orunsubstituted cycloalkyl, R^(7CF)-substituted or unsubstitutedheterocycloalkyl, R^(7CF)-substituted or unsubstituted aryl, orR^(7CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(7C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(7CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(7CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(7CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(7CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(7CF)-substituted or unsubstitutedphenyl, or R^(7CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(7B) and R^(7C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(7CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(7C)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(7D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(7DF)-substituted or unsubstituted alkyl,R^(7DF)-substituted or unsubstituted heteroalkyl, R^(7DF)-substituted orunsubstituted cycloalkyl, R^(7DF)-substituted or unsubstitutedheterocycloalkyl, R^(7DF)-substituted or unsubstituted aryl, orR^(7DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(7D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(7DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(7DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(7DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(7DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(7DF)-substituted or unsubstitutedphenyl, or R^(7DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(8A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(1AF)-substituted or unsubstituted alkyl,R^(8AF)-substituted or unsubstituted heteroalkyl, R^(8AF)-substituted orunsubstituted cycloalkyl, R^(8AF)-substituted or unsubstitutedheterocycloalkyl, R^(8AF)-substituted or unsubstituted aryl, orR^(8AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(8A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(8AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(8AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(8AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(8AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(8AF)-substituted or unsubstitutedphenyl, or R^(8AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(8B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(8BF)-substituted or unsubstituted alkyl,R^(8BF)-substituted or unsubstituted heteroalkyl, R^(8BF)-substituted orunsubstituted cycloalkyl, R^(8BF)-substituted or unsubstitutedheterocycloalkyl, R^(8BF)-substituted or unsubstituted aryl, orR^(8AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(8B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(8BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(8BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(8BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(8BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(8BF)-substituted or unsubstitutedphenyl, or R^(8BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(8C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(8CF)-substituted or unsubstituted alkyl,R^(8CF)-substituted or unsubstituted heteroalkyl, R^(8CF)-substituted orunsubstituted cycloalkyl, R^(8CF)-substituted or unsubstitutedheterocycloalkyl, R^(8CF)-substituted or unsubstituted aryl, orR^(8CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(8C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(8CF)-substituted or unsubstituted C₁-C₆ alkyl, R^(8CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(8CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(8CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(8CF)-substituted or unsubstitutedphenyl, or R^(8CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(8B) and R^(8C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(8CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(8CF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(8D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(8DF)-substituted or unsubstituted alkyl,R^(8DF)-substituted or unsubstituted heteroalkyl, R^(8DF)-substituted orunsubstituted cycloalkyl, R^(8DF)-substituted or unsubstitutedheterocycloalkyl, R^(8DF)-substituted or unsubstituted aryl, orR^(8DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(8D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(8DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(8DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(8DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(8DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(8DF)-substituted or unsubstitutedphenyl, or R^(8DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(9A) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(9AF)-substituted or unsubstituted alkyl,R^(9AF)-substituted or unsubstituted heteroalkyl, R^(9AF)-substituted orunsubstituted cycloalkyl, R^(9AF)-substituted or unsubstitutedheterocycloalkyl, R^(9AF)-substituted or unsubstituted aryl, orR^(9AF)-substituted or unsubstituted heteroaryl. In embodiments, R^(9A)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(9AF)-substituted or unsubstituted C₁-C₆ alkyl, R^(9AF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(9AF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(9AF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(9AF)-substituted or unsubstitutedphenyl, or R^(9AF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(9B) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(9BF)-substituted or unsubstituted alkyl,R^(9BF)-substituted or unsubstituted heteroalkyl, R^(9BF)-substituted orunsubstituted cycloalkyl, R^(9BF)-substituted or unsubstitutedheterocycloalkyl, R^(9BF)-substituted or unsubstituted aryl, orR^(9BF)-substituted or unsubstituted heteroaryl. In embodiments, R^(9B)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(9BF)-substituted or unsubstituted C₁-C₆ alkyl, R^(9BF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(9BF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(9BF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(9BF)-substituted or unsubstitutedphenyl, or R^(9BF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

In embodiments, R^(9C) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(9CF)-substituted or unsubstituted alkyl,R^(9CF)-substituted or unsubstituted heteroalkyl, R^(9CF)-substituted orunsubstituted cycloalkyl, R^(9CF)-substituted or unsubstitutedheterocycloalkyl, R^(9CF)-substituted or unsubstituted aryl, orR^(9CF)-substituted or unsubstituted heteroaryl. In embodiments, R^(9C)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R⁹CF-substituted or unsubstituted C₁-C₆ alkyl, R^(9CF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(9CF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(9CF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(9CF)-substituted or unsubstitutedphenyl, or R^(9CF)-substituted or unsubstituted 5 to 6 memberedheteroaryl. R^(9B) and R^(9C) bonded to the same nitrogen atom mayoptionally be joined to form a R^(9CF)-substituted or unsubstituted 3 to6 membered heterocycloalkyl or R^(9CF)-substituted or unsubstituted 5 to6 membered heteroaryl.

In embodiments, R^(9D) is independently hydrogen, halogen, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂,—OCHBr₂, —OCHI₂, R^(9DF)-substituted or unsubstituted alkyl,R^(9DF)-substituted or unsubstituted heteroalkyl, R^(9DF)-substituted orunsubstituted cycloalkyl, R^(9DF)-substituted or unsubstitutedheterocycloalkyl, R^(9DF)-substituted or unsubstituted aryl, orR^(9DF)-substituted or unsubstituted heteroaryl. In embodiments, R^(9D)is independently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,R^(9DF)-substituted or unsubstituted C₁-C₆ alkyl, R^(9DF)-substituted orunsubstituted 2 to 6 membered heteroalkyl, R^(9DF)-substituted orunsubstituted C₃-C₆ cycloalkyl, R^(9DF)-substituted or unsubstituted 3to 6 membered heterocycloalkyl, R^(9DF)-substituted or unsubstitutedphenyl, or R^(9DF)-substituted or unsubstituted 5 to 6 memberedheteroaryl.

R^(1F), R^(2F), R^(3F), R^(4F), R^(5F), R^(6F), R^(7F), R^(8F), R^(9F),R^(1AF), R^(1BF), R^(1CF), R^(1DF), R^(2AF), R^(2BF), R^(2CF), R^(2DF),R^(3AF), R^(3BF), R^(3CF), R^(3DF), R^(4AF), R^(4BF), R^(4CF), R^(4DF),R^(5AF), R^(5BF), R^(5CF), R^(5DF), R^(6AF), R^(6BF), R^(6CF), R^(6DF),R^(7AF), R^(7BF), R^(7CF), R^(7DF), R^(8AF), R^(8BF), R^(8AF), R^(8DF),R^(9AF), R^(9BF), R^(9CF) and R^(9DF) are independently oxo, halogen,—CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H,—NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R^(1F),R^(2F), R^(3F), R^(4F), R^(5F), R^(6F), R^(7F), R^(8F), R^(9F), R^(1AF),R^(1BF), R^(1CF), R^(1DF), R^(2AF), R^(2BF), R^(2CF), R^(2DF), R^(3AF),R^(3BF), R^(3CF), R^(3DF), R^(4AF), R^(4BF), R^(4CF), R^(4DF), R^(5AF),R^(5BF), R^(5CF), R^(5DF), R^(6AF), R^(6BF), R^(6CF), R^(6DF), R^(7AF),R^(7BF), R^(7CF), R^(7DF), R^(8AF), R^(8BF), R^(8CF), R^(8DF), R^(9AF),R^(9BF), R^(9CF) and R^(9DF) are independently oxo, halogen, —CF₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,—NHOH, —OCF₃, —OCHF₂, unsubstituted C₁-C₆ alkyl, unsubstituted 2 to 6membered heteroalkyl, unsubstituted C₃-C₆ cycloalkyl, unsubstituted 3 to6 membered heterocycloalkyl, unsubstituted phenyl, or unsubstituted 5 to6 membered heteroaryl.

In some embodiments, a compound as described herein may include multipleinstances of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, m1, m2, m6, m7, m8, m9,n1, n2, n6, n7, n8, n9, v1, v2, v6, v7, v8, v9, and/or other variables.In such embodiments, each variable may optional be different and beappropriately labeled to distinguish each group for greater clarity. Forexample, where each R¹, R², R³, R¹, R⁵, R⁶, R⁷, R¹, R⁹, m1, m2, m6, m7,m8, m9, n1, n2, n6, n7, n8, n9, v1, v2, v6, v7, v8 and/or v9 isdifferent, they may be referred to, for example, as R^(1.1), R^(1.2),R^(1.3), R^(1.4), R^(1.5), R^(1.6), R^(1.7), R^(2.1), R^(2.2), R^(2.3),R^(2.4), R^(2.5), R^(2.6), R^(2.7), R^(3.1), R^(3.2), R^(3.3), R^(3.4),R^(3.5), R^(3.6), R^(3.7), R^(4.1), R^(4.2), R^(7.1), R^(7.2), R^(7.3),R^(7.4), R^(7.5), R^(7.6), R^(7.7), R^(8.1), R^(8.2), R^(8.3), R^(8.4),R^(8.5), R^(8.6), R^(8.7), R^(9.1), R^(9.2), R^(9.3), R^(9.4), R^(9.5),R^(9.6), R^(9.7), m1¹, m1², m1³, m1⁴, m1⁵, m1⁶, m1⁷, m2¹, m2², m2³, m2⁴,m2⁵, m2⁶, m2⁷, m6¹, m6², m6³, m6⁴, m6⁵, m6⁶, m6⁷, m7¹, m7², m7³, m7⁴,m7⁵, m7⁶, m7⁷, m8¹, m8², m8³, m8⁴, m8⁵, m8⁶, m8⁷, m9¹, m9², m9³, m9⁴,m9⁵, m9⁶, m9⁷, n1¹, n1², n1³, n1⁴, n1⁵, n1⁶, n1⁷, n2¹, n2², n2³, n2⁴,n2⁵, n2⁶, n2⁷, n6¹, n6², n6³, n6⁴, n6⁵, n6⁶, n6⁷, n7¹, n7², n7³, n7⁴,n7⁵, n7⁶, n7⁷, n8¹, n8², n8³, n8⁴, n8⁵, n8⁶, n8⁷, n9¹, n9², n9³, n9⁴,n9⁵, n9⁶, n9⁷, v1¹, v1², v1³, v1⁴, v1⁵, v1⁶, v1⁷, v2¹, v2², v2³, v2⁴,v2⁵, v2⁶, v2⁷, v6¹, v6², v6³, v6⁴, v6⁵, v6⁶, v6⁷, v7¹, v7², v7³, v7⁴,v7⁵, v7⁶, v7⁷, v8¹, v8², v8³, v8⁴, v8⁵, v8⁶, v8⁷, v9¹, v9², v9³, v9⁴,v9⁵, v9⁶, v9⁷, respectively, wherein the definition of R¹ is assumed byR^(1.1), R^(1.2), R^(1.3), R^(1.4), R^(1.5), R^(1.6), R^(1.7), thedefinition of R² is assumed by R^(2.1), R^(2.2), R^(2.3), R^(2.4),R^(2.5), R^(2.6), R^(2.7), the definition of R³ is assumed by R^(3.1),R^(3.2), R^(3.3), R^(3.4), R^(3.5), R^(3.6), R^(3.7), the definition ofR is assumed by R^(4.1), R^(4.2), R^(4.3), R^(4.4), R^(4.5), R^(4.6),R^(4.7), the definition of R⁵ is assumed by R^(5.1), R^(5.2), R^(5.3),R^(5.4), R^(5.4), R^(5.6), R^(5.7), the definition of R⁶ is assumed byR⁶, R^(6.2), R^(6.3), R^(6.4), R^(6.5), R^(6.6), R^(6.7) the definitionof R⁷ is assumed by R^(7.1), R^(7.2), R^(7.3), R^(7.4), R^(7.5),R^(7.6), R^(7.7) the definition of R⁸ is assumed by R^(8.1), R^(8.2),R^(8.3), R^(8.4), R^(8.5), R^(8.6), R^(8.7), the definition of R⁹ isassumed by R^(9.1), R^(9.2), R^(9.3), R^(9.4), R^(9.5), R^(9.6),R^(9.7), the definition of m1¹ is assumed by m1¹, m1², m1³, m1⁴, m1⁵,m1⁶, m1⁷, the definition of m2 is assumed by m2¹, m2², m2³, m2, m2⁵,m2⁶, m2⁷, the definition of m6 is assumed by m6¹, m6², m6³, m6⁴, m6⁵,m6⁶, m6⁶, the definition of m7 is assumed by m7¹, m7², m7³, m7⁴, m7⁵,m7⁶, m7⁶, the definition of m8 is assumed by m8¹, m8², m8³, m8⁴, m8⁵,m8⁶, m8⁷, the definition of m9 is assumed by m9¹, m9², m9³, m9⁴, m9⁵,m9⁶, m9⁷, the definition of n1 is assumed by n1¹, n1², n1³, n1⁴, n1⁵,n1⁶, n1⁷, the definition of n2 is assumed by n2¹, n2², n2³, n2⁴, n2⁵,n2⁶, n2⁷, is assumed by n3¹, n3², n3³, n3⁴, n3⁵, n3⁶, n3⁷, thedefinition of n4 is assumed by n4¹, n4², n4³, n4⁴, n4⁵, n4⁶, n4⁷, thedefinition of n5 is assumed by n5¹, n5², n5³, n5⁴, n5⁵, n5⁶, n5⁷, thedefinition of n6 is assumed by n6¹, n6², n6³, n6⁴, n6⁵, n6⁶, n6⁷, thedefinition of n7 is assumed by n7¹, n7², n7³, n7⁴, n7⁵, n7⁶, n7⁷, thedefinition of n8 is assumed by n8¹, n8², n8³, n8⁴, n8⁵, n8⁶, n8⁷, thedefinition of n9 is assumed by n9¹, n9², n9³, n9⁴, n9⁵, n9⁶, n9⁷, thedefinition of v1 is assumed by v1¹, v1², v1³, v1⁴, v1⁵, v1⁶, v1⁷, thedefinition of v2 is assumed by v2¹, v2², v2³, v2⁴, v2⁵, v2⁶, v2⁷, thedefinition of v3 is assumed by v3¹, v3², v3³, v3⁴, v3⁵, v3⁶, v3⁷, thedefinition of v4 is assumed by v4¹, v4², v4³, v4⁴, v4⁵, v4⁶, v4⁷, thedefinition of v5 is assumed by v5¹, v5², v5³, v5⁴, v5⁵, v5⁶, v5⁷, thedefinition of v6 is assumed by v6¹, v6², v6³, v6⁴, v6⁵, v6⁶, v6⁷, thedefinition of v7 is assumed by v7¹, v7², v7³, v7⁴, v7⁵, v7⁶, v7⁷, thedefinition of v8 is assumed by v8¹, v8², v8³, v8⁴, v8⁵, v8⁶, v8⁷ and thedefinition of v9 is assumed by v9¹, v9², v9³, v9⁴, v9⁵, v9⁶, v9⁷. Thevariables used within a definition of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, m1, m2, m3, m4, m5, m6, m7, m8, m9, n1, n2, n3, n4, n5, n6, n7, n8,n9, v1, v2, v3, v4, v5, v6, v7, v8, v9 and/or other variables thatappear at multiple instances and are different may similarly beappropriately labeled to distinguish each group for greater clarity.

In embodiments, R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ arejoined to form, together with the atoms to which they are attached,substituted or unsubstituted (e.g. C₃-C₆) cycloalkyl, substituted orunsubstituted (e.g. 3 to 6 membered) heterocycloalkyl, substituted orunsubstituted (e.g. phenyl) aryl, or substituted or unsubstituted (e.g.5 to 6 membered) heteroaryl.

In embodiments, R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ arejoined to substituted or unsubstituted (e.g. 3 to 6 membered)heterocycloalkyl, substituted or unsubstituted (e.g. phenyl) aryl, orsubstituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl. Inembodiments, R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ are joinedto substituted or unsubstituted (e.g. phenyl) aryl, or substituted orunsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R¹ andR⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ are joined to substituted orunsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R¹ andR⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸ and R⁹ are joined to form, together withthe atoms to which they are attached, a substituted or unsubstitutedpyrazolyl, oxazolyl, or thiazolyl. In embodiments, R¹ and R⁶, R⁶ and R⁷,R¹ and R⁹, or R⁸ and R⁹ are joined to form, together with the atoms towhich they are attached, unsubstituted pyrazolyl, oxazolyl, orthiazolyl.

In embodiments, R¹ and R⁶ are joined to form, together with the atoms towhich they are attached, R^(1E)-substituted or unsubstituted (e.g.C₃-C₆) cycloalkyl, R^(1E)-substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl, R^(1E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(1E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R¹ and R⁶ are joined to form,together with the atoms to which they are attached, R^(1E)-substitutedor unsubstituted (e.g. 3 to 6 membered) heterocycloalkyl,R^(1E)-substituted or unsubstituted (e.g. phenyl) aryl, orR^(1E)-substituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl.In embodiments, R¹ and R⁶ are joined to form, together with the atoms towhich they are attached, R^(1E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(1E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R¹ and R⁶ are joined to form,together with the atoms to which they are attached, R^(1E)-substitutedor unsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R¹and R⁶ are joined to form, together with the atoms to which they areattached, R^(1E)-substituted or unsubstituted pyrazolyl, oxazolyl, orthiazolyl. In embodiments, R¹ and R⁶ are joined to form, together withthe atoms to which they are attached, unsubstituted pyrazolyl, oxazolyl,or thiazolyl. In embodiments, the compound is represented formula IB¹,IC¹, ID¹, or IE¹:

In embodiments, the compound is represented formula IB1A, IC1A, IB1A′,IC1A′, ID1A, or IE1A:

In embodiments, R⁶ and R⁷ are joined to form, together with the atoms towhich they are attached, R^(6E)-substituted or unsubstituted (e.g.C₃-C₆) cycloalkyl, R^(6E)-substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl, R^(6E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(6E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R⁶ and R⁷ are joined to form,together with the atoms to which they are attached, R^(6E)-substitutedor unsubstituted (e.g. 3 to 6 membered) heterocycloalkyl,R^(6E)-substituted or unsubstituted (e.g. phenyl) aryl, orR^(6E)-substituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl.In embodiments, R⁶ and R⁷ are joined to form, together with the atoms towhich they are attached, R^(6E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(6E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R⁶ and R⁷ are joined to form,together with the atoms to which they are attached, R^(6E)-substitutedor unsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R⁶and R⁷ are joined to form, together with the atoms to which they areattached, R^(6E)-substituted or unsubstituted pyrazolyl, oxazolyl, orthiazolyl. In embodiments, R⁶ and R⁷ are joined to form, together withthe atoms to which they are attached, unsubstituted pyrazolyl, oxazolyl,or thiazolyl. In embodiments, the compound is represented formula IB2,IC2, ID2, or IE2:

In embodiments, the compound is represented formula IB2A, IC2A, IB2A′,IC2A′, ID2A, or IE2A.

In embodiments, R¹ and R⁹ are joined to form, together with the atoms towhich they are attached, R^(9E)-substituted or unsubstituted (e.g.C₃-C₆) cycloalkyl, R^(9E)-substituted or unsubstituted (e.g. 3 to 6membered) heterocycloalkyl, R^(9E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(9E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R¹ and R⁹ are joined to form,together with the atoms to which they are attached, R^(9E)-substitutedor unsubstituted (e.g. 3 to 6 membered) heterocycloalkyl,R^(9E)-substituted or unsubstituted (e.g. phenyl) aryl, orR^(9E)-substituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl.In embodiments, R¹ and R⁹ are joined to form, together with the atoms towhich they are attached, R^(9E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(9E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R¹ and R⁹ are joined to form,together with the atoms to which they are attached, R^(9E)-substitutedor unsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R¹and R⁹ are joined to form, together with the atoms to which they areattached, R^(9E)-substituted or unsubstituted pyrazolyl, oxazolyl, orthiazolyl. In embodiments, R¹ and R⁹ are joined to form, together withthe atoms to which they are attached, unsubstituted pyrazolyl, oxazolyl,or thiazolyl. In embodiments, the compound is represented formula IB3,IC3, ID3, or IE3:

In embodiments, the compound is represented formula IB3A, IC3A, IB3A′,IC3A′, ID3A, or IE3A:

In embodiments, R¹ and R⁹ are joined to form, together with the atoms towhich they are attached, RE-substituted or unsubstituted (e.g. C₃-C₆)cycloalkyl, R^(8E)-substituted or unsubstituted (e.g. 3 to 6 membered)heterocycloalkyl, R^(8E)-substituted or unsubstituted (e.g. phenyl)aryl, or R^(8E)-substituted or unsubstituted (e.g. 5 to 6 membered)heteroaryl. In embodiments, R¹ and R⁹ are joined to form, together withthe atoms to which they are attached, R^(8E)-substituted orunsubstituted (e.g. 3 to 6 membered) heterocycloalkyl,R^(8E)-substituted or unsubstituted (e.g. phenyl) aryl, orR^(8E)-substituted or unsubstituted (e.g. 5 to 6 membered) heteroaryl.In embodiments, R¹ and R⁹ are joined to form, together with the atoms towhich they are attached, R^(8E)-substituted or unsubstituted (e.g.phenyl) aryl, or R^(8E)-substituted or unsubstituted (e.g. 5 to 6membered) heteroaryl. In embodiments, R¹ and R⁹ are joined to form,together with the atoms to which they are attached, R^(8E)-substitutedor unsubstituted (e.g. 5 to 6 membered) heteroaryl. In embodiments, R¹and R⁹ are joined to form, together with the atoms to which they areattached, R^(8E)-substituted or unsubstituted pyrazolyl, oxazolyl, orthiazolyl. In embodiments, R⁸ and R⁹ are joined to form, together withthe atoms to which they are attached, unsubstituted pyrazolyl, oxazolyl,or thiazolyl. In embodiments, the compound is represented formula IB4,IC4, ID4, or IE4:

In embodiments, the compound is represented formula IB4A IC4A IB4A′,IC4A′, ID4 or IE4:

R¹, R², R³, R⁴, R⁵, R⁷, R⁸, and/or R⁹ are as described herein. Inembodiments, R² is C₂-C₄ haloalkyl, R³ and R⁴ are independentlyhydrogen, methyl or ethyl; R⁵, R⁷, R⁸ and R⁹ are hydrogen. Inembodiments, R² is C₂-C₆ alkyl substituted with at least one fluorine;R³ are R⁴ are independently methyl or ethyl; R⁵, R⁷, R⁸ and R⁹ arehydrogen. In embodiments, R² is —CH(CF₃)₂ or —CH₂(CF₂)₂H.

In embodiments, the compound is not

In embodiments, the compound is:

In embodiments, the compound is formula I,

or a pharmaceutically acceptable salt thereof. In embodiments, X is —O—.In embodiments, R² is C₂-C₄ haloalkyl. In embodiments, R³ is hydrogen.In embodiments, R¹ is —CH₃ or —CH₂CH₃. In embodiments, R¹, R⁶, R⁷, R⁸and R⁹ are hydrogen. In embodiments, R² is substituted with at leastfour fluorines. In embodiments, R² is substituted with four fluorines.In embodiments, R² is —CH₂(CF₂)₂H. In embodiments, X is —O—; R² is—CH₂(CF₂)₂H; R³ is hydrogen; R is —CH₃ or —CH₂CH₃; and R¹, R⁶, R⁷, R⁸and R⁹ are hydrogen. In embodiments, the compound is

In embodiments, the compound is a compound described herein (e.g., in anaspect, embodiment, example, table, figure, scheme, appendix, or claim).

II. Pharmaceutical Compositions

Also provided herein are pharmaceutical formulations. In embodiments,the pharmaceutical formulations include the compounds described above(including all embodiments thereof) (e.g. formulae I, IA, IB1, IC1, ID1,IE1, IB1A, IC1A, IB1A′, IC1A′, ID1A, IE1A, IB2, IC2, ID2, IE2, IB2A,IC2A, B2A′, IC2A′, ID2A, IE2A, IB3A, IC3A, IB3A′, IC3A′, ID3A, IE3A,IB4A, IC4A, IB4A′, IC4A′, ID4A and IE4A) and a pharmaceuticallyacceptable excipient. In one aspect is a pharmaceutical composition thatincludes a compound described herein and a pharmaceutically acceptableexcipient.

In embodiments, the pharmaceutical composition comprises apharmaceutically acceptable excipient, and a compound of Formula I:

R⁴ (I), or a pharmaceutically acceptable salt thereof,

X, R¹, R², R³, R⁴, R⁵, R⁷, R⁸, and R⁹ are as described herein.

In embodiments, X is —O— or —S—. In embodiments, X is NH. Inembodiments, X is —O—.

In embodiment, R² is —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃ or C₂-C₄haloalkyl. In embodiments, R³, R⁴ and R⁵ are independently hydrogen, orsubstituted or unsubstituted C₂-C₆ alkyl. In embodiments, R¹ ishydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂,—C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CF₃—CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CH₂F, —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂or substituted or unsubstituted alkyl; and R⁹ is hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R^(1D)R^(6D), R^(7D), R^(8D) andR^(9D) are independent hydrogen or methyl. In embodiments, R⁷ and R⁸ areindependently hydrogen.

In embodiments, the compound is formula IA:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are as described herein.

In embodiments, R² is —CX^(2.1) ₃, —(CH₂)_(n2)CX^(2.1) ₃, or C₂-C₄haloalkyl. In embodiments, R³, R⁴ and R⁵ are independently hydrogen,substituted or unsubstituted alkyl. In embodiments, R¹ is hydrogen,halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂, —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl. In embodiments, R⁷ and R⁸ are independentlyhydrogen. In embodiments, R³ and R⁴ substituted or unsubstituted alkyl;R⁵ are independently hydrogen; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl; wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independently hydrogen or methyl, and X^(1.1), X^(2.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently are —F. In embodiments,R³ and R⁴ are independently methyl or ethyl. In embodiments, R¹ ishydrogen, halogen, —NO₂, or —⁻COOH; R⁵ is hydrogen; R⁶ is hydrogen,halogen —NO₂, or —COOH; R⁷ and R⁸ are independently hydrogen; and R⁹ ishydrogen, halogen NO₂, or —COOH. In embodiments, wherein R³ and R⁴ areindependently hydrogen, methyl or ethyl. In embodiments, wherein R² isC₂-C₄ alkyl substituted with at least one fluorine. In embodiments, R¹,R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

In embodiments, R¹ and R⁶ are joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted pyrazolyl,oxazolyl, or thiazolyl; R² is C₂-C₄ haloalkyl; R³ and R¹ areindependently hydrogen, methyl or ethyl. In embodiments, the compound isrepresented with Formula IB, IC, ID, or IE:

R⁸ and R⁹ are independently hydrogen. In embodiments, R² is —CH(CF₃)₂ or—CH₂(CF₂)₂H.

In embodiments, the pharmaceutical composition comprises a compoundselected from:

In embodiments of the pharmaceutical compositions, the compound, orpharmaceutically acceptable salt thereof, is included in atherapeutically effective amount.

1. Formulations

The pharmaceutical composition may be prepared and administered in awide variety of dosage formulations. Compounds described may beadministered orally, rectally, or by injection (e.g. intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally).

For preparing pharmaceutical compositions from compounds describedherein, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier may be one or more substance that may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material.

In powders, the carrier may be a finely divided solid in a mixture withthe finely divided active component. In tablets, the active componentmay be mixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% to 70% of the activecompound. Suitable carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

In embodiments, liquid form preparation or solid form preparationcomprising the compounds as described herein may not include DMSO in theformulations. In embodiments, the liquid form preparation may notinclude solvating agent such as DMSO and the compound included in thepreparation may maintain the same or substantially same amounts ofdissolved or suspended compounds, for example, greater than about 50 wt%, 55 wt %, 60 wt %, 65 wt %, 70 wt %, 75 wt %, 80 wt %, 85 wt %, 90 wt%, 95 wt %, or 99 wt % of the dissolved or suspended compounds which arebeing prepared in the liquid form preparation including DMSO. In someembodiments, the liquid form preparation or solid form preparation notincluding DMSO may exhibit the same or substantially same penetration ofthe compound across mucus, mucous membrane or skin, for example, greaterthan about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% ofpenetration of the compounds which are prepared in the formulationincluding DMSO, when administered topically, transmucousally ortransdermally.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60, and 80; Pluronic F-68,F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Suchco-solvents are typically employed at a level between about 0.01% andabout 2% by weight. Viscosity greater than that of simple aqueoussolutions may be desirable to decrease variability in dispensing theformulations, to decrease physical separation of components of asuspension or emulsion of formulation, and/or otherwise to improve theformulation. Such viscosity building agents include, for example,polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxy propyl cellulose, chondroitin sulfate and salts thereof,hyaluronic acid and salts thereof, and combinations of the foregoing.Such agents are typically employed at a level between about 0.01% andabout 2% by weight.

The pharmaceutical compositions may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharides,and finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760. The entire contents of these patents areincorporated herein by reference in their entirety for all purposes.

The pharmaceutical composition may be intended for intravenous use. Thepharmaceutically acceptable excipient can include buffers to adjust thepH to a desirable range for intravenous use. Many buffers includingsalts of inorganic acids such as phosphate, borate, and sulfate areknown.

2. Effective Dosages

The pharmaceutical composition may include compositions wherein theactive ingredient is contained in a therapeutically effective amount,i.e., in an amount effective to achieve its intended purpose. The actualamount effective for a particular application will depend, inter alia,on the condition being treated.

The dosage and frequency (single or multiple doses) of compoundsadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated; presence of other diseases or otherhealth-related problems; kind of concurrent treatment; and complicationsfrom any disease or treatment regimen. Other therapeutic regimens oragents can be used in conjunction with the methods and compoundsdisclosed herein.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring response of theconstipation or dry eye to the treatment and adjusting the dosageupwards or downwards, as described above.

Dosages may be varied depending upon the requirements of the subject andthe compound being employed. The dose administered to a subject, in thecontext of the pharmaceutical compositions presented herein, should besufficient to effect a beneficial therapeutic response in the subjectover time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects. Generally,treatment is initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compounds effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

3. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p.l, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

When parenteral application is needed or desired, particularly suitableadmixtures for the compounds included in the pharmaceutical compositionmay be injectable, sterile solutions, oily or aqueous solutions, as wellas suspensions, emulsions, or implants, including suppositories.

In particular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and thelike. Ampoules are convenient unit dosages. Pharmaceutical admixturessuitable for use in the pharmaceutical compositions presented herein mayinclude those described, for example, in Pharmaceutical Sciences (17thEd., Mack Pub. Co., Easton, Pa.) and WO 96/05309, the teachings of bothof which are hereby incorporated by reference.

III. Methods of Activating

Further provided herein are methods of activating a Cystic FibrosisTransmembrane Conductance Regulator (CFTR). The method includescontacting the CFTR with an effective amount of a compound describedherein, thereby activating the CFTR. In one aspect, the method includescontacting CFTR with an effective amount of the compound of Formula I:

or a pharmaceutically acceptable salt thereof. X, R¹, R², R³, R⁴, R⁵,R⁶, R⁷, R⁸ and R⁹ are described herein.

In embodiments, the method includes contacting CFTR with an effectiveamount of the compound as described herein thereby activating CFTR. Thecontacting may be performed in vitro. The contacting may be performed invivo.

IV. Methods of Treating

Further provided herein are methods of treating a disease or disorder ina subject in need thereof by administering to said subject an effectiveamount of a compound as described herein (e.g. a compound of formula I):

or a pharmaceutically acceptable salt thereof. X, R¹, R², R³, R⁴, R⁵,R⁶, R⁷, R⁸ and R⁹ are described herein.

In one aspect is a method of treating constipation in a subject in needthereof, the method including administering to the subject an effectiveamount described compound as described herein (e.g. a compound offormula I). In another aspect, is a method of treating a dry eyedisorder in a subject in need thereof, the method includingadministering to the subject an effective amount of a compound asdescribed herein (e.g. a compound of formula I). In yet another aspect,is a method of increasing lacrimation in a subject in need thereof, themethod including administering to the subject an effective amount acompound as described herein (e.g. a compound of formula I).

In one aspect, provided is a method of treating a cholestatic liverdisease in a subject in need thereof, including administering to thesubject an effective amount a compound as described herein (e.g. acompound of formula I). In another aspect, provided is a method oftreating a pulmonary disease or disorder in a subject in need thereof,including administering to the subject an effective amount of asdescribed herein (e.g. a compound of formula I). In embodiments, thepulmonary disease or disorder is chronic obstructive pulmonary disease(e.g. bronchitis, asthma, cigarette smoke-induced lung dysfunction).

Other Aspects

Provided herein, in another aspect, are compositions and methods oftreating a disease. The following definitions and embodiments apply toonly to the compounds of formula (pI), this section (i.e. section V) andembodiments P1 to P28 listed herein.

For purposes of this section, the term “alkyl” refers to and includeslinear or branched univalent hydrocarbon structures and combinationthereof, which may be fully saturated, mono- or polyunsaturated, havingthe number of carbon atoms designated (i.e., C₁-C₁₀ means one to tencarbons). Particular alkyl groups are those having 1 to 20 carbon atoms(a “C₁-C₂₀ alkyl”). More particular alkyl groups are those having 1 to 8carbon atoms (a “C₁-C₈ alkyl”), 3 to 8 carbon atoms (a “C₃-C₈ alkyl”), 1to 6 carbon atoms (a “C₁-C₆ alkyl”), 1 to 5 carbon atoms (a “C₁-C₅alkyl”), or 1 to 4 carbon atoms (a “C₁-C₄ alkyl”). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl,n-heptyl, n-octyl, and the like. An unsaturated alkyl group is onehaving one or more double bonds or triple bonds. Examples of unsaturatedalkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. Examples of saturated C₁-C₄ alkyl include methyl (CH₃), ethyl(C₂H₅), propyl (C₃H₇) and butyl (C₄H₉). Examples of saturated C₁-C₆alkyl include methyl (CH₃), ethyl (C₂H₅), propyl (C₃H₇), butyl (C₄H₉),pentyl (C₅H₁₁) and hexyl (C₆H₁₃).

An alkyl group may be substituted (i.e., one or more hydrogen atoms arereplaced with univalent or divalent radicals) with one moresubstituents, such as radicals described herein, for example, fluoro,chloro, bromo, iodo, hydroxyl, alkoxy, thio, amino, acylamino,alkoxycarbonylamido, carboxyl, acyl, alkoxycarbonyl, sulfonyl,cycloalkyl, aryl, heterocyclyl and heteroaryl, and other functionalgroups known in the art. A “perfluoroalkyl” refers to an alkyl groupwhere every hydrogen atom is replaced with a fluorine atom. Examples ofsaturated C₁-C₆ perfluroalkyl include trifluoromethyl (CF₃),pentafluoroethyl (C₂F₅), heptafluoropropyl (C₃F₇), nonafluorobutyl(C₄F₉), undecafluoropentyl (C₅F₁₁) and tridecafluorohexyl (C₆F₁₃).

For purposes of this section, the term “cycloalkyl” refers to andincludes cyclic univalent hydrocarbon structures, which may be fullysaturated, mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e., C₁-C₁₀ means one to ten carbons). Cycloalkyl canconsist of one ring, such as cyclohexyl, or multiple rings, such asadamantly, but excludes aryl groups. A cycloalkyl comprising more thanone ring may be fused, spiro or bridged, or combinations thereof. Apreferred cycloalkyl is a cyclic hydrocarbon having from 3 to 13 annularcarbon atoms. A more preferred cycloalkyl is a cyclic hydrocarbon havingfrom 3 to 8 annular carbon atoms (a “C₃-C₈ cycloalkyl”). Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,norbornyl, and the like.

For purposes of this section, the term “heterocycle” or “heterocyclyl”refers to a saturated or an unsaturated non-aromatic group having from 1to 10 annular carbon atoms and from 1 to 4 annular heteroatoms, such asnitrogen, sulfur or oxygen, and the like, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heterocyclyl group may have a single ring ormultiple condensed rings, but excludes heteroaryl groups. A heterocyclecomprising more than one ring may be fused, spiro or bridged, or anycombination thereof. In fused ring systems, one or more of the fusedrings can be aryl or heteroaryl. Examples of hetercyclyl groups include,but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl,piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl,tetrahydrofuranyl, tetrahydrothiophenyl,2,3-dihydrobenzo[b]thiophen-2-yl, 4-amino-2-oxopyrimidin-1 (2H)-yl, andthe like.

For purposes of this section, the term “aryl” refers to and includespolyunsaturated aromatic hydrocarbon substituents. Aryl may containadditional fused rings (e.g., from 1 to 3 rings), including additionallyfused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. In onevariation, the aryl group contains from 6 to 14 annular carbon atoms.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, biphenyl, and the like.

For purposes of this section, the term “heteroaryl” refers to andincludes unsaturated aromatic cyclic groups having from 1 to 10 annularcarbon atoms and at least one annular heteroatom, including but notlimited to heteroatoms such as nitrogen, oxygen and sulfur, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule at an annular carbon or annularheteroatom. Heteroaryl may contain additional fused rings (e.g., from 1to 3 rings), including additionally fused aryl, heteroaryl, cycloalkyl,and/or heterocyclyl rings. Examples of heteroaryl groups include, butare not limited to, pyridyl, pyrimidyl, thiophenyl, furanyl, thiazolyl,and the like.

Cycloalkyl, aryl, heterocyclyl and heteroaryl groups as referred towithin this section may also be substituted with one or moresubstituents, such as radicals detailed herein, for example, fluoro,chloro, bromo, iodo, hydroxyl, alkoxy, thio, amino, acylamino,alkoxycarbonylamido, carboxyl, acyl, alkoxycarbonyl, sulfonyl, alkyl,cycloalkyl, aryl, hetercyclyl and herteroaryl, and other functionalgroups known in the art.

For purposes of this section, the term “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical formulation, otherthan an active ingredient, which is nontoxic to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative, such as those known inthe art, for example, described in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

As used in this section, “treatment” or “treating” is an approach forobtaining beneficial or desired results including and preferablyclinical results. For example, beneficial or desired clinical resultsinclude, but are not limited to, one or more of the following:decreasing symptoms resulting from the disease, increasing the qualityof life of those suffering from the disease, decreasing the dose ofother medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of individuals.

As used in this section, the phrase “delaying development of a disease”means to defer, hinder, slow, retard, stabilize, and/or postponedevelopment of the disease (such as constipation, dry eye, pulmonarydisease or disorder, lung disease or liver disease). This delay can beof varying lengths of time, depending on the history of the diseaseand/or individual being treated. As is evident to one skilled in theart, a sufficient or significant delay can, in effect, encompassprevention, in that the individual does not develop the disease.

As used in this section, an “effective dosage” or “effective amount” ofdrug, compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the onset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such asdecreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, enhancingeffect of another medication such as via targeting, delaying theprogression of the disease, and/or prolonging survival. An effectivedosage can be administered in one or more administrations. For purposesof this section, an effective dosage of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used in this section, “in conjunction with” refers to administrationof one treatment modality in addition to another treatment modality. Assuch, “in conjunction with” refers to administration of one treatmentmodality before, during or after administration of the other treatmentmodality to the individual.

Unless clearly indicated otherwise, for purposes of this section, theterm “individual” as used herein refers to a mammal, including but notlimited to, bovine, horse, feline, rabbit, canine, rodent, or primate(e.g., human). In some embodiments, an individual is a human. In someembodiments, an individual is a non-human primate such as chimpanzeesand other apes and monkey species. In some embodiments, an individual isa farm animal such as cattle, horses, sheep, goats and swine; pets suchas rabbits, dogs and cats; laboratory animals including rodents, such asrats, mice, and guinea pigs; and the like. The aspects described in thissection may find use in both human medicine and in the veterinarycontext.

As used in this section and in the appended embodiments P1-P25, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly indicates otherwise.

It is understood that aspect and variations of the aspects described inthis section include “consisting” and/or “consisting essentially of”aspects and variations.

Constipation therapy includes laxatives that increase stool bulk, suchas soluble fiber; create an osmotic load, such as polyethylene glycol;or stimulate intestinal contraction, such as the diphenylmethanes. Thereare also surface laxatives that soften stool such as docusate sodium andprobiotics such as Lactobacillus paracasei [3]. The FDA-approved druglinaclotide, a peptide agonist of the guanylate cyclase C receptor, actsby inhibiting visceral pain, stimulating intestinal motility, andincreasing intestinal secretion [4, 5]. A second approved drug,lubiprostone, a prostaglandin E analog, is thought to activate aputative enterocyte ClC-2 channel [6], though the mechanistic data areless clear. Despite the wide range of therapeutic options, there is acontinued need for safe and effective drugs to treat constipation.

Without wishing to be bound by theory, in embodiments of this section,activation of the cystic fibrosis transmembrane regulator (CFTR)chloride channel drives fluid secretion in the intestine, whichmaintains lubrication of luminal contents. It is hypothesized thatdirect activation of CFTR may cause fluid secretion and reverseexcessive dehydration of stool found in constipation.

Intestinal fluid secretion involves active Cl⁻ secretion across theenterocyte epithelium through the basolateral membrane Na⁺/K⁺/2Cl⁻cotransporter (NKCC1) and the luminal membrane cystic fibrosistransmembrane regulator (CFTR) Cl⁻ channel and Ca²⁺-activated Cl⁻channel (CaCC). The electrochemical and osmotic forces created by Cl⁻secretion drive Na⁺ and water secretion [7]. In cholera and Traveler'sdiarrhea CFTR is strongly activated by bacterial enterotoxins throughelevation of intracellular cyclic nucleotides [8, 9]. CFTR is anattractive target to increase intestinal fluid secretion in constipationas it is robustly expressed throughout the intestine and its activationstrongly increases intestinal fluid secretion. An activator targetingCFTR directly is unlikely to produce the massive, uncontrolledintestinal fluid secretion seen in cholera because the enterotoxins incholera act irreversibly to produce sustained elevation of cytoplasmiccAMP, which not only activates CFTR but also basolateral K⁺ channels,which increase the electrochemical driving force for Cl⁻ secretion;cholera enterotoxins also inhibit the luminal NHE3 Na⁺/H⁺ exchangerinvolved in intestinal fluid absorption [10, 11].

Motivated by these considerations and the continuing need for safe andeffective drug therapy of constipation, the identification andcharacterization of a nanomolar-potency, CFTR-targeted small-moleculeactivators with pro-secretory action in intestine and efficacy inconstipation are reported herein.

By high-throughput screening a nanomolar-affinity, small-molecule CFTRactivator, CFTR_(act)-J027 was identified and demonstrated to havepro-secretory action in mouse intestine and efficacy in normalizingstool output in a loperamide-induced mouse model of constipation.Constipation remains a significant clinical problem in outpatient andhospitalized settings. Opioid-induced constipation is a common adverseeffect in patients after surgery, undergoing chemotherapy and withchronic pain.

CFTR-targeted activation adds to the various mechanisms of action ofanti-constipation therapeutics. It is notable that pure CFTR activationis able to produce a robust Cl⁻ current and fluid secretion response inthe intestine, without causing global elevation of cyclic nucleotideconcentration, direct stimulation of intestinal contractility, oralteration of intestinal fluid absorption. Linaclotide, a peptideagonist of the guanylate cyclase C receptor that increases intestinalcell cGMP concentration. Linaclotide inhibits activation of colonicsensory neurons and activates motor neurons, which reduces pain andincreases intestinal smooth muscle contraction; in addition, elevationin cGMP concentration in enterocytes may activate CFTR and have apro-secretory action [4, 5]. A second approved drug, the prostaglandin Eanalog lubiprostone, is thought to activate a putative enterocyte ClC-2channel [6], though the mechanistic data are less clear. Compared withthese drugs, a pure CFTR activator has a single, well-validatedmechanism of action and does not produce a global cyclic nucleotideresponse in multiple cell types. Of note, linaclotide and lubiprostoneshowed limited efficacy in clinical trials. Linaclotide was effective in˜20% of chronic constipation patients of whom ˜5% also responded toplacebo [15], and lubiprostone was effective in ˜13% of IBS-C patientsof whom ˜7% responded to placebo [16]. Based on our mouse data showingsubstantially greater efficacy of CFTR_(act)-J027 compared tosupramaximal doses of linaclotide or lubiprostone, we speculate thatCFTR activators may have greater efficacy in clinical trials.

CFTR_(act)-J027 is more potent for activation of wildtype CFTR thanVX-770 (ivacaftor), the FDA-approved drug for treatment of cysticfibrosis (CF) caused by certain CFTR gating mutations. In FRT cellsexpressing wild-type CFTR, short-circuit current measurement showednearly full activation of CFTR by CFTR_(act)-J027 at 3 μM whereas VX-770maximally activated CFTR by only 15%. However, CFTR_(act)-J027 wassubstantially less potent than ivacaftor as a ‘potentiator’ of defectivechloride channel gating of the most common CF-causing mutation, ΔF508,which is not unexpected, as potentiator efficacy in CF ismutation-specific. In addition to its potential therapeutic utility forconstipation, a small-molecule activator of wildtype CFTR may be usefulfor treatment of chronic obstructive pulmonary disease and bronchitis,asthma, cigarette smoke-induced lung dysfunction, dry eye andcholestatic liver disease [17-19].

Substituted quinoxalinones were reported as selective antagonists of themembrane efflux transporter multiple-drug-resistance protein 1 [20].Quinoxalinones have also been reported to show anti-diabetic activity bystimulating insulin secretion in pancreatic INS-1 cells [21], andinhibitory activity against serine proteases for potential therapy ofthrombotic disorders [22]. Recently, quinoxalinones have been reportedto inhibit aldose reductase [23]. These reports suggest that thequinoxalinone scaffold has drug-like properties. Synthetically,quinoxalinone can be prepared in one to four steps from commerciallyavailable starting materials [24], which allows facile synthesis oftargeted analogs.

In addition to compound-specific off-target actions, the potentialside-effects profile of a CFTR activator could include pro-secretoryactivity in the airway/lungs and various glandular and other epithelia.Off-target effects for constipation therapy could be limited by oraladministration of a CFTR activator with limited intestinal absorptionand/or rapid systemic clearance to minimize systemic exposure.CFTR_(act)-J027 when administered orally at a high dose (10 mg/kg)showed very low bioavailability with blood levels well below the EC₅₀for CFTR activation, which may be due to first-pass effect as evidencedits rapid in vitro metabolism in liver microsomes. CFTR_(act)-J027 didnot show significant in vitro cytotoxicity at a concentration of 25μM, >100-fold greater than its EC₅₀ for CFTR activation, or in vivotoxicity in mice in a 7-day study at a maximal efficacious dose thatnormalized stool output in the loperamide model of constipation. Thepotentially most significant off-target action, stimulation oflung/airway fluid secretion, was not seen as evidenced by normal lungwater content in the 7-day treated mice. These limited toxicity studiesoffer proof of concept for application of a CFTR activator inconstipation.

In summary, the data presented herein demonstrate the pro-secretoryaction of a CFTR activator in mouse intestine for use in treatment ofvarious types of constipation, which could include opioid-inducedconstipation, chronic idiopathic constipation, and irritable bowelsyndrome with constipation predominance.

Dry eye disorders, including Sjögren's syndrome, constitute a commonproblem in the aging population with limited effective therapeuticoptions available. The cAMP-activated Cl⁻ channel CFTR (cystic fibrosistransmembrane conductance regulator) is a major pro-secretory chloridechannel at the ocular surface. It was investigated whether compoundsthat target CFTR can correct the abnormal tear film in dry eye.Small-molecule activators of human wild-type CFTR identified byhigh-throughput screening were evaluated in cell culture and in vivoassays to select compounds that stimulate Cl⁻-driven fluid secretionacross the ocular surface in mice. An aminophenyl-1,3,5-triazine,CFTR_(act)-K089, fully activated CFTR in cell cultures with EC₅₀˜250 nMand produced a ˜8.5 mV hyperpolarization in ocular surface potentialdifference. When delivered topically, CFTR_(act)-K089 doubled basal tearsecretion for four hours and had no effect in CF mice. CFTR_(act)-K089showed sustained tear film bioavailability without detectable systemicabsorption. In a mouse model of aqueous-deficient dry eye produced bylacrimal gland excision, topical administration of 0.1 nmolCFTR_(act)-K089 three times daily restored tear secretion to basallevels and fully prevented the corneal epithelial disruption seen invehicle-treated controls. The data presented herein demonstratepotential utility of CFTR-targeted activators as a novel pro-secretorytreatment for dry eye.

Ninety-four percent of surveyed ophthalmologists believe that additionaltreatments are needed for moderate-to-severe dry eye (7).

The ocular surface is a collection of anatomically continuous epithelialand glandular tissues that are functionally linked to maintain the tearfilm (8). While lacrimation contributes the bulk of reflex tearing, thecomea and conjunctiva regulate basal tear volume and composition. Theprincipal determinants of water movement across the ocular surface intothe tear film include apical chloride (Cl⁻) secretion through cAMP- andcalcium (Ca²⁺)-dependent Cl⁻ transporters, and sodium (Na⁺) absorptionlargely though the epithelial Na⁺ channel (ENaC).

With regard to pro-secretory candidates for dry eye therapy, an ENaCinhibitor, P321, has recently entered phase 1/2 studies (9). Diquafosol,a UTP analog that targets surface epithelial P2Y₂ receptors andstimulates Cl⁻ and mucin secretion by Ca²⁺ signaling (10), is approvedfor dry eye in Japan (11, 12) but failed phase III trials in the UnitedStates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is acAMP-activated Cl⁻ channel expressed in some secretory epithelial cells,including those in cornea and conjunctiva (14-16). We found substantialcapacity for active CFTR-facilitated Cl⁻ at the ocular surface in mice(21, 22), as subsequently shown in rat conjunctiva (23), providing arational basis for investigation of CFTR activators as a pro-secretorystrategy for dry eye. The only clinically approved CFTR activator,VX-770 (ivacaftor), is indicated for potentiating the channel gating ofcertain CFTR mutants causing CF, but only weakly activates wild-typeCFTR (24, 25).

Novel small-molecule activators of wild-type CFTR identified byhigh-throughput screening as potential topical therapy for dry eye wereevaluated to demonstrate efficacy of newly identified CFTR activator(s)in a mouse model of dry eye.

The potential utility of small-molecule activators of CFTR for dry eyetherapy was investigated. After several prior development failures, dryeye remains an unmet need in ocular disease. It was hypothesized thatCFTR-targeted pro-secretory compounds could normalize tear film volumeand ocular surface properties in dry eye (21, 22). In dry eye disorders,tear film hyperosmolarity stimulates pro-inflammatory signaling,secretion of cytokines and metalloproteinases, and disruption of cornealepithelial cell integrity (35-38). By minimizing tear filmhyperosmolarity, CFTR activation is predicted to prevent thesedownstream ocular surface changes.

Small-molecule CFTR activators were identified by high-throughputscreening that produced sustained Cl⁻-driven aqueous fluid secretionacross the ocular surface by a mechanism involving direct CFTRactivation rather than upstream cAMP signaling. The rationale to choosecompounds that activate CFTR directly was to minimize potentialoff-target effects of generalized cAMP stimulation and to reduce thelikelihood of tachyphylaxis for compounds targeting signaling receptors.These compounds had low-nanomolar EC₅₀ for activation of human CFTR invitro and produced full activation at higher concentrations. LargeCFTR_(act)-dependent PD hyperpolarizations and tear hypersecretion weredemonstrated in mice. Substantial compound activities in mice and humanswill facilitate translation of data here to humans.

It was found that CFTR_(act)-K089 restored tear secretion and preventedepithelial disruption in an experimental mouse model of lacrimalinsufficiency. CFTR activators may be particularly suited for disordersof the lacrimal gland, such as primary Sjögren's syndrome, bystimulating fluid transport across the intact corneal and conjunctivalepithelia. CFTR activators probably exert their major pro-secretoryeffect at the ocular surface, although there is indirect for CFTRexpression and function in lacrimal gland (39-42). Direct stimulation oflacrimal secretion is unlikely in the studies here because of minimalcompound penetration to lacrimal tissues following topical delivery, andthe demonstrated compound efficacy in a model of lacrimal insufficiency.At the ocular surface, the conjunctiva probably contributes the bulk offluid secretion given its much larger surface area compared to cornea(43).

Alternative pro-secretory therapies targeting different ocular surfaceion channels have been considered. The only FDA-approved CFTR activator,VX-770, was developed as a “potentiator” to treat CF by correcting thechannel gating of certain CFTR mutations (44). However, VX-770 showedrelatively little activity against wild-type CFTR in cell cultures andin mice in vivo. Chronic application of VX-770 may also diminish CFTRfunctional expression (24) and cause cataracts (seen in juvenile rats;ref. 42), which is likely an off-target effect because CFTR is notexpressed in lens.

An indirect agonist of Ca²⁺-activated Cl⁻ channel(s), diquafosol,augments both aqueous and mucin secretion. However, diquafosol failedphase III trials, likely due to transient induced Ca²⁺ elevation and Cl⁻channel activation, producing minimal net fluid secretion. CFTRactivators, which produce sustained tear fluid secretion, overcome thislimitation. CFTR-K089 and CFTR_(act)-J027 showed favorablepharmacodynamics and could be conveniently administered topicallyseveral times daily in a standard ophthalmic formulation.

The data presented herein show that CFTR activation alone facilitatessustained outward Cl⁻ flux and fluid secretion, suggesting that basal K⁺conductance, without augmented cyclic nucleotide or Ca²⁺ signaling, issufficient to support ocular surface fluid transport. Still, thepotential synergy of a CFTR agonist and a K⁺ channel activator or anENaC inhibitor could be explored to further increase tear secretion fordry eye therapy.

The efficacy of CFTR_(act)-K089 in a clinically relevant mouse model ofaqueous-deficient dry eye disease was demonstrated for topical,pro-secretory CFTR activator therapy to restore basal tear secretion andprevent ocular surface pathology. Compared with immunosuppressiveapproaches, CFTR activation has the advantage of addressing an earlyevent in dry eye pathogenesis. Our data thus support the developmentpotential of CFTR activators as first-in-class dry eye therapy.

Examples herein provide further disclosure on aspects and embodiments ofthis section.

Although the foregoing section has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itis apparent to those skilled in the art that certain minor changes andmodifications will be practiced in light of the above teaching.

Therefore, the description and examples should not be construed aslimiting the scope of any invention described herein.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

Embodiments contemplated herein include embodiments P1 to P28 following.

Embodiment P1

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient, and a compound of Formula I:

pharmaceutically acceptable salt thereof, wherein: X is O, NH or S; n1is independently an integer from 0 to 4; m1 and v1 are eachindependently 1 or 2; R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1)₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C),—NHNR^(1B)R^(1C), —ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C),—NHC(O)NR^(1B)R^(1C), —N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D),—C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R² ishydrogen, —CX^(2.1) ₃, CHX^(2.1) ₂, —(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl or haloalkyl; R³ is hydrogen,—COR^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A),—C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl; R⁴ ishydrogen, —COR^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(8B)R^(8C), —C(O)OR^(5D),—SO₂R^(5A)C(O)NR^(8B)R^(5C), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, whereinR³ and R⁴ may optionally be joined to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m1), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n1)R^(7A), —SO_(v1)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m1), —NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D),—C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C),—NR^(7B)C(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n1)R^(8A),—SO_(v1)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m1),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n), R^(9A), —SO_(v1)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m1), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR, —NR^(9B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(1A).R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B),R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C),R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D),R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) areindependently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C)R^(3B), R^(3C),R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B),R^(8C), R^(9B) and R^(9C) substituents bonded to the same nitrogen atommay optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(3.1), X^(4.1), X^(5.1), X^(6.1), X^(7.1), X^(8.1)and X^(9.1) are independently —Cl, —Br, —I or —F; with the proviso thatwhen X is O; R² is —(CH₂)_(n1)CX^(2.1) ₃; n1 is 1; X^(2.1) is fluorine;R³ is hydrogen; and R is methyl, then R⁶ is not —NO₂; and when X is O;R² is —(CH₂)_(n1)CX^(2.1) ₃; n1 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R is methyl, then R⁹ is not —NO₂.

Embodiment P2

The pharmaceutical composition of embodiment P1, wherein: X is O; R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—N(O)_(m1), —C(O)R^(8D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, or substituted or unsubstitutedheteroaryl; R³, R⁴ and R⁵ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform a substituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —N(O)_(m1), —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, orsubstituted or unsubstituted heteroaryl; R⁷ is hydrogen, halogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m1), —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl; R⁸ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —N(O)_(m1), —C(O)R^(8D),—C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl; and R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m1), —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,or substituted or unsubstituted heteroaryl.

Embodiment P3

The pharmaceutical composition of embodiment P1, wherein: X is O; R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n1)CX^(2.1) ₃, substituted or unsubstituted alkyl,substituted or unsubstituted aryl or haloalkyl; R³, R⁴ and R⁵ areindependently hydrogen, substituted or unsubstituted alkyl orsubstituted or unsubstituted aryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —N(O)_(m1), —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—N(O)_(m1), —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ orsubstituted or unsubstituted alkyl; R¹ is hydrogen, halogen, —CX^(8.1)₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —N(O)_(m1), —C(O)R^(8D),—C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl; and R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m1), —C(O)R^(9D), —C(O)OR^(9D),—OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted or unsubstituted alkyl.

Embodiment P4

The pharmaceutical composition of embodiment P1, wherein: X is O; R¹ ishydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN,—N(O)_(m1), —C(O)R^(1D), —C(O)OR^(8D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(6.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, substituted alkyl, substituted orunsubstituted aryl or haloalkyl; R³ and Rare independently hydrogen,substituted or unsubstituted alkyl or substituted or unsubstituted aryl;R⁵ is hydrogen or substituted or unsubstituted alkyl; R⁶ is hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO, —C(O)R^(6D),—C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted orunsubstituted alkyl; R⁷ is hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃,—CHF₂, —CH₂F, —CN, —N(O)_(m1), —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂ or substituted or unsubstituted alkyl; R⁸ is hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —N(O)_(m1),—C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted orunsubstituted alkyl; and R⁹ is hydrogen, halogen, —CF₃, —CCl₃, —CBr₃,—CI₃, —CHF₂, —CH₂F, —CN, —N(O)_(m1), —C(O)R^(9D), —C(O)OR^(9D),—OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted or unsubstituted alkyl.

Embodiment P5

The pharmaceutical composition of embodiment P4, wherein at least two ofR¹, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

Embodiment P6

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient, and a compound of Formula IA:

or a pharmaceutically acceptable salt thereof, wherein: n1 is an integerfrom 0 to 4; m1 and v1 are each independently 1 or 2; R¹ is hydrogen,halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —SO_(n1)R^(1A),—SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C), —ONR^(1B)R^(1C),—NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C), —N(O)_(m1),—NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A),—NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D),—NR^(1B)OR^(1D), —OCX¹ ₃, —OCHX¹ ₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R² ishydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂, —(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl or haloalkyl; R³ is hydrogen,—COR^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A),—C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl; R⁴ ishydrogen, —COR^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(1D), —SO₂R^(5A),—C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m1), —NR^(6B)R^(6C), —C(O)R⁶, —C(O)OR^(6D), —C(O)NR^(6B)R^(6C),—OR^(6A), —NRSO₂R^(6A), —NR^(6B)C(O)R^(6D), —NR^(6B)(O)OR^(6D),—NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁷ is hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n1)R^(7A), —SO_(v1)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m1), —NR^(7B)R^(7C), —C(O)R⁷, —C(O)OR^(7D),—C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C),—NR^(7B)C(O)OR^(7D), —NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁸ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n), R^(8A),—SO_(v1)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m1),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8B),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n1)R^(9A), —SO_(v1)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m1), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D), —NR^(8B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(1A),R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B),R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C),R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D),R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) areindependently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B),R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C),R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to the samenitrogen atom may optionally be joined to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl; and X^(1.1), X^(2.1), X^(3.1), X^(4.1), X^(5.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently —Cl, —Br, —I or —F; withthe proviso that when X is O; R² is —(CH₂)_(n1)CX^(1.1) ₃; n1 is 1;X^(1.1) is fluorine; R³ is hydrogen; and R is methyl, then R⁶ is not—NO₂.

Embodiment P7

The pharmaceutical composition of embodiment P6, wherein R² is hydrogen,—CX^(2.1) ₃, —CHX^(2.1) ₂, —OR^(2A), unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl.

Embodiment P8

The pharmaceutical composition of embodiment P7, wherein: R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R³, R⁴ and R⁵ are independentlyhydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —N(O)_(m1), —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m1),—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —N(O)_(m1), —C(O)R^(8D), —C(O)OR^(8D), —OCX^(1.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m1),—C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl.

Embodiment P9

The pharmaceutical composition of embodiment P6, wherein R⁶ is hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n1)R^(6A),—SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C), —ONR^(6B)R^(6C),—NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C), —NO, —NR^(6B)R^(6C),—C(O)R^(6A), —C(O)OR^(6A), —C(O)NR^(6B)R^(6A), —NR^(6B)SO₂R^(6A),—NR^(6C)(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.

Embodiment P10

The pharmaceutical composition of embodiment P9, wherein: R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R³, R⁴ and R⁵ are independentlyhydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —N(O)_(m1), —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m1),—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R⁸ is hydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —N(O)_(m1), —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m1),—C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl.

Embodiment P11

The pharmaceutical composition of embodiment P6, wherein R³ is—COR^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A),—C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl.

Embodiment P12

The pharmaceutical composition of embodiment P11, wherein: R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—N(O)_(m1), —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R³, R⁴ and R⁵ are independentlyhydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —N(O)_(m1), —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —N(O)_(m1),—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R⁸ is hydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X—^(8.1), —CN, —N(O)_(m1), —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —N(O)_(m1),—C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl.

Embodiment P13

The pharmaceutical composition of embodiment P6, wherein at least two ofR¹, R⁶, R⁷, R⁸ and R⁹ are independently hydrogen.

Embodiment P14

The pharmaceutical composition of embodiment P13, wherein: R¹ ishydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂,—NO, —C(O)R^(1A), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n1)CX^(2.1) ₃, substituted alkyl, substituted orunsubstituted aryl or haloalkyl; R³ and R⁴ are independently hydrogen,substituted or unsubstituted alkyl or substituted or unsubstituted aryl;R⁵ is hydrogen or substituted or unsubstituted alkyl; R⁶ is hydrogen,halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO, —C(O)R^(6D),—C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX⁶² or substituted or unsubstitutedalkyl; R⁷ is hydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1),—CN, —NO, —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX⁷² orsubstituted or unsubstituted alkyl; R¹ is hydrogen, halogen, —CX^(8.1)₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —NO, —C(O)R^(8D), —C(O)OR^(8D),—OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; andR⁹ is hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—NO, —C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ orsubstituted or unsubstituted alkyl.

Embodiment P15

The pharmaceutical composition of embodiment P14, wherein: R¹ is ahydrogen, halogen or —NO₂; R² is —(CH₂)_(n1)CX^(2.1) ₃ or substitutedalkyl; and R⁵ is hydrogen.

Embodiment P16

The pharmaceutical composition of embodiment P15, wherein R³ and R⁴ areindependently hydrogen, methyl or ethyl.

Embodiment P17

The pharmaceutical composition of embodiment P16, wherein R² is alkylsubstituted with at least one fluorine.

Embodiment P18

The pharmaceutical composition of embodiment P17, wherein R⁶, R⁷, R⁸ andR⁹ are independently hydrogen and R¹ is —NO₂.

Embodiment P19

The pharmaceutical composition of embodiment P17, wherein R¹, R⁶, R⁷, R⁸and R⁹ are independently hydrogen.

Embodiment P20

A method of treating constipation, comprising administering to a subjectin need thereof a therapeutically effective amount of a compound ofstructural Formula

or a pharmaceutically acceptable salt thereof, wherein: X is O, NH or S;n1 is an integer from 0 to 4; m1 and v1 are each independently 1 or 2;R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX¹ ₃, —OCHX¹ ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂,—(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl; R³ is hydrogen, —COR^(3D), —C(O)NHNR^(3B)R^(3C),—C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C)-substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; R⁴ is hydrogen, —COR^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A),—C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m1), —NR^(6B)R^(6C), —C(O)R^(6D), —C(O)OR^(6D),—C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A), —NR^(6B)C(O)R^(6D),—NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁷ is hydrogen, halogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n1)R^(7A),—SO_(v1)NR^(7B)R^(7C), —NHNR^(7B)R^(7C), —ONR^(7B)R^(7C),—NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C), —N(O)_(m1),—NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A),—NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D),—NR^(8B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁸ is hydrogen, halogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n1)R^(8A), —SO_(v1)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m1), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8D),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n1)R^(9A), —SO_(v1)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(9B)R^(9C), —NHC(O)NHNR^(8B)R^(9C), —NHC(O)NR^(8B)R^(9C),—N(O)_(m1), —NR^(8B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D),—C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D),—NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D),R^(2A), R^(2B), R^(2C)R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A),R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B),R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C),R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) andR^(9C) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(3.1),X^(4.1), X^(5.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F.

Embodiment P21

The method of embodiment P20, further comprising administering to thesubject an anti-constipation agent.

Embodiment P22

The method of embodiment P20, wherein the compound is administeredorally.

Embodiment P23

The method of embodiment P20, wherein the constipation is opioid-inducedconstipation, chronic idiopathic constipation or irritable bowelsyndrome with constipation predominance.

Embodiment P24

A method of treating a dry eye disorder, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof structural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: X is O, NH or S;n1 is an integer from 0 to 4; m1 and v1 are each independently 1 or 2;R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX¹ ₃, —OCHX¹ ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂,—(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl; R³ is hydrogen, —COR^(3D), —C(O)NHNR^(3B)R^(3C),—C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; R⁴ is hydrogen, —COR^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A),—C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m1), —NR^(6B)R^(6C), —C(O)R⁶, —C(O)OR⁶, —C(O)NR^(6B)R^(6C),—OR^(6A), —NR^(6B)SO₂R^(6A), —NR^(6C)(O)R^(6D), —NR^(6C)(O)OR^(6D),—NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁷ is hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n1)R^(7A), —SO_(v1)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m1), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(8D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D), —NR^(8B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹ ishydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN,—SO_(n1)R^(8A), —SO_(v1)NR^(8B)R^(8C), —NHNR^(8B)R^(8C),—ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C),—N(O)_(m1), —NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D),—C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(1B)SO₂R^(8A), —NR^(8B)C(O)R^(8D),—NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n1)R^(9A),—SO_(v1)NR^(9B)R^(9C), —NHNR^(9B)R^(9C), —ONR^(9B)R^(9C),—NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O)_(m1),—NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D), R^(2A),R^(2B), R^(2C), R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B),R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C),R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D),R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6A), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B)and R^(9C) substituents bonded to the same nitrogen atom may optionallybe joined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(3.1),X^(4.1), X^(5.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F.

Embodiment P25

The method of embodiment P24, wherein the dry eye disorder is a lacrimalgland disorder.

Embodiment P26

The method of embodiment P24, further comprising administering to thesubject an anti-dry eye agent.

Embodiment P27

A method of increasing lacrimation, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundof structural Formula

or a pharmaceutically acceptable salt thereof, wherein: X is O, NH or S;n1 is an integer from 0 to 4; m1 and v1 are each independently 1 or 2;R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX¹ ₃, —OCHX¹ ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂,—(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl; R³ is hydrogen, —COR^(3D), —C(O)NHNR^(3B)R^(3C),—C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; R⁴ is hydrogen, —COR^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A),—C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m1), —NR^(6B)R^(6C), —C(O)R⁶, —C(O)OR⁶, —C(O)NR^(6B)R^(6C),—OR^(6A), —NR^(6B)SO₂R^(6A), —NR^(6C)(O)R^(6D), —NR^(6C)(O)OR^(6D),—NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁷ is hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n1)R^(7A), —SO_(v1)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m1), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7A)C(O)R^(7C), —NR^(7B)BC(O)OR^(7D), —NR^(8B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹ ishydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN,—SO_(n1)R^(8A), —SO_(v1)NR^(8B)R^(8C), —NHNR^(8B)R^(8C),—ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C),—N(O)_(m1), —NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D),—C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D),—NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n1)R^(9A),—SO_(v1)NR^(9B)R^(9C), —NHNR^(9B)R^(9C), —ONR^(9B)R^(9C),—NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O)_(m1),—NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(8B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D), R^(2A),R^(2B), R^(2C)R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B),R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C),R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D),R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) andR^(9C) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(3.1),X^(4.1), X^(5.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F.

Embodiment P28

A method of activating Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR), comprising contacting CFTR with a compound ofstructural Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: X is O, NH or S;n1 is an integer from 0 to 4; m1 and v1 are each independently 1 or 2;R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX¹ ₃, —OCHX¹ ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂,—(CH₂)_(n1)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl orhaloalkyl; R³ is hydrogen, —COR^(3D), —C(O)NHNR^(3B)R^(3C),—C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C)-substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; R⁴ is hydrogen, —COR^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁵ ishydrogen, —COR^(5D), —C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A),—C(O)NR^(5B)R^(5C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; R⁶ ishydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—SO_(n1)R^(6A), —SO_(v1)NR^(6B)R^(6C), —NHNR^(6B)R^(6C),—ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C), —NHC(O)NR^(6B)R^(6C),—N(O)_(m1), —NR^(6B)R^(6C), —C(O)R^(6D), —C(O)OR⁶, —C(O)NR^(6B)R^(6C),—OR^(6A), —NR^(1B)SO₂R^(6A), —NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D),—NR^(6B)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁷ is hydrogen, halogen, —CX^(7.1) ₃,—CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n1)R^(7A), —SO_(v1)NR^(7B)R^(7C),—NHNR^(7B)R^(7C), —ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C),—NHC(O)NR^(7B)R^(7C), —N(O)_(m1), —NR^(7B)R^(7C), —C(O)R^(7D),—C(O)OR^(8D), —C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A),—NR^(7A)C(O)R^(7C), —NR^(7B)BC(O)OR^(7D), —NR^(8B)OR^(7D), —OCX^(7.1) ₃,—OCHX^(7.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹ ishydrogen, halogen, —CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN,—SO_(n1)R^(8A), —SO_(v1)NR^(8B)R^(8C), —NHNR^(8B)R^(8C),—ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C),—N(O)_(m1), —NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D),—C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D),—NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n1)R^(9A),—SO_(v1)NR^(9B)R^(9C), —NHNR^(9B)R^(9C), —ONR^(9B)R^(9C),—NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O)_(m1),—NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D), R^(2A),R^(2B), R^(2C)R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B),R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C),R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D),R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) andR^(9C) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl orsubstituted or unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(3.1),X^(4.1), X^(5.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F.

Further embodiments contemplated herein include embodiment 1 tofollowing.

Embodiment 1

A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O— or —S—;n1, n2, n6, n7, n8, and n9 are independently an integer from 0 to 4; m1,m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently 1 or 2; R¹is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C), —ONR^(1B)R^(1C),—NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C), —N(O)_(m1),—NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A),—NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D)NR^(1B)C(O)OR^(1D),—NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃, —CH₃,—(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted or unsubstituted C₂-C₈alkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl or C₂-C₄ haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; R⁴ ishydrogen, —C(O)R^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl, wherein R³ and R⁴ are optionally be joined to form, togetherwith the atoms to which they are attached, a substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R⁵ is hydrogen, —C(O)R^(5D), —C(O)NHNR^(5B)R^(5C),—C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —SO_(n)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO_(n7)R^(7A),—SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C), —ONR^(7B)R^(7C),—NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C), —N(O)_(m7),—NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A),—NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7C),—NR^(8B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1)2,—CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R″, —C(O)OR′,—C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D),—NR^(8A)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁹ is hydrogen, halogen,—CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n1)R^(9A),—SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C), —ONR^(9B)R^(9C),—NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O), —NR^(9B)R^(9C),—C(O)R^(9D), —C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹are optionally joined to form, together with the atoms to which they areattached, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D),R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A),R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B),R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C),R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B),R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C),R^(9B) and R^(9C) substituents bonded to the same nitrogen atom mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(6.1), X^(7.1),X^(8.1) and X^(9.1) are independently —Cl, —Br, —I or —F; with theproviso that when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R¹ is substituted orunsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂; and when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X²¹ is fluorine; R³ is hydrogen; and R¹is substituted or unsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂, withthe proviso that when X is —O—; R² is —CH₂(CF₂)₂H; R³ is hydrogen; and Ris unsubstituted C₁-C₃ alkyl, then R¹ is not —NO₂; with the proviso thatwhen X is —O—; R² is —CH(CF₃)₂; R³ and R⁴ are independentlyunsubstituted C₁-C₃ alkyl, then R¹ is not hydrogen; and with the provisothat when X is —O— and R² is methyl substituted with cycloalkyl, then R³and R are hydrogen.

Embodiment 2

The compound of embodiment 1, wherein R² is —CX^(2.1) ₃,—(CH₂)_(n2)CX^(2.1) ₃ or C₂-C₄ haloalkyl.

Embodiment 3

The compound of embodiment 1 or 2, wherein R³, R⁴ and R⁵ areindependently hydrogen, or substituted or unsubstituted C₁-C₄ alkyl.

Embodiment 4

The compound of embodiment 1, 2 or 3, wherein: R¹ is hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CH₂F, —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂or substituted or unsubstituted alkyl; and R⁹ is hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl, wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independent hydrogen or methyl.

Embodiment 5

The compound of embodiment 4, wherein R⁷ and R⁸ are independentlyhydrogen.

Embodiment 6

The compound of embodiment 1, wherein the compound is represented byFormula IA:

or a pharmaceutically acceptable salt thereof.

Embodiment 7

The compound of embodiment 6, wherein R² is —CX^(2.1) ₃,—(CH₂)_(n2)CX^(2.1) ₃, or C₂-C₄ haloalkyl.

Embodiment 8

The compound of embodiment 6 or 7, wherein R³, R⁴ and R⁵ areindependently hydrogen, substituted or unsubstituted alkyl.

Embodiment 9

The compound of embodiment 6, 7 or 8, wherein: R¹ is hydrogen, halogen,—CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂, —C(O)R^(1D),—C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl.

Embodiment 10

The compound of embodiment 9, wherein R⁷ and R⁸ are independentlyhydrogen.

Embodiment 11

The compound of embodiment 6 or 7, wherein: R³ and R⁴ are independentlymethyl or ethyl; R⁵ are hydrogen; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl; wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independently hydrogen or methyl, and X^(1.1), X^(2.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently are —F.

Embodiment 12

The compound of embodiment 11, wherein: R¹ is hydrogen, halogen, —NO₂,or —⁻COOH; R⁵ is hydrogen; R⁶ is hydrogen, halogen —NO₂, or —COOH; R⁷and R⁸ are independently hydrogen; and R⁹ is hydrogen, halogen NO₂, or—COOH.

Embodiment 13

The compound of embodiment 6 or 7, wherein R³ and R⁴ are independentlyhydrogen, methyl or ethyl.

Embodiment 14

The compound of embodiment 4, wherein R² is C₂-C₄ alkyl substituted withat least one fluorine.

Embodiment 15

The compound of embodiment 6, 7 or 14, wherein R¹, R⁵, R⁶, R⁷, R⁸ and R⁹are hydrogen.

Embodiment 16

The compound of embodiment 6 or 7, wherein R¹ and R⁶ are joined to form,together with the atoms to which they are attached, a substituted orunsubstituted pyrazolyl, oxazolyl, or thiazolyl.

Embodiment 17

The compound of embodiment 16, wherein R³ and R⁴ are independentlyhydrogen, methyl or ethyl.

Embodiment 18

The compound of embodiment 16, wherein the compound is represented withFormula IB, IC, ID, or IE:

Embodiment 19

The compound of embodiment 18, wherein R⁷, R⁸ and R⁹ are hydrogen.

Embodiment 20

The compound of embodiment 14, 15, 16, 18 or 19, wherein R² is —CH(CF₃)₂or —CH₂(CF₂)₂H.

Embodiment 21

The compound of embodiment 1, wherein the compound is selected from:

Embodiment 22

A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—; R² isC₂-C₄ haloalkyl; R³ is hydrogen; R is —CH₃ or —CH₂CH₃; and R¹, R⁶, R⁷,R⁸ and R⁹ are hydrogen.

Embodiment 23

The compound of embodiment 22, wherein R² is substituted with at leastfour fluorines.

Embodiment 24

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient, and a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O— or —S—;n1, n2, n6, n7, n8, and n9 are independently an integer from 0 to 4; m1,m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently 1 or 2; R¹is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN,—SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D)NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R² ishydrogen, —CX^(2.1) ₃, CH₃, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substitutedor unsubstituted C₂-C₈ alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; R⁴ ishydrogen, —C(O)R^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl, wherein R³ and R⁴ are optionally be joined to form, togetherwith the atoms to which they are attached, a substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R⁵ is hydrogen, —C(O)R^(5D), —C(O)NHNR^(5B)R^(5C),—C(O)OR^(8D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, wherein R³ and R⁴ may optionally be joined toform, together with the atoms to which they are attached, a substitutedor unsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂,—CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6.1), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6C)(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —SO₇R^(7A),—SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C), —ONR^(7B)R^(7C),—NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C), —N(O)_(m7),—NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D), —C(O)NR^(7B)R^(7C), —OR^(7A),—NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n1)R^(9A), —SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C), —N(O),—NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A),—NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹are optionally joined to form, together with the atoms to which they areattached, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D),R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A),R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B),R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(1B), R^(8C),R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃,—OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B), R^(3C), R^(4B),R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C),R^(9B) and R^(9C) substituents bonded to the same nitrogen atom mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(6.1), X^(7.1),X^(8.1) and X^(9.1) are independently —Cl, —Br, —I or —F; with theproviso that when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R¹ is substituted orunsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂; and when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X²¹ is fluorine; R³ is hydrogen; and Ris substituted or unsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂, andwith the proviso that when X is —O— and R² is methyl substituted withcycloalkyl, then R³ and R⁴ are hydrogen.

Embodiment 25

The pharmaceutical composition of embodiment 24, wherein R² is —CX^(2.1)₃, —(CH₂a)_(n2)CX^(2.1) ₃ or C₂-C₄ haloalkyl.

Embodiment 26

The pharmaceutical composition of embodiment 24 or 25, wherein R³, R⁴and R⁵ are independently hydrogen, or substituted or unsubstituted C₁-C₄alkyl.

Embodiment 27

The pharmaceutical composition of embodiment 24, 25 or 26, wherein: R¹is hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CBr₃, —CI₃, —CHF₂, —CH₂F,—CN, —NO₂, —C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ orsubstituted or unsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1)₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D),—OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ ishydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂,—C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R¹ is hydrogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂,—CH₂F, —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂or substituted or unsubstituted alkyl; and R⁹ is hydrogen, halogen,—CF₃, —CCl₃, —CBr₃, —CI₃, —CHF₂, —CH₂F, —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl, wherein R^(1D), R^(6D), R^(7D)R^(8D) and R^(9D) areindependent hydrogen or methyl.

Embodiment 28

The pharmaceutical composition of embodiment 27, wherein R⁷ and R⁸ areindependently hydrogen.

Embodiment 29

The pharmaceutical composition of embodiment 24, wherein the compound isrepresented by Formula IA:

or a pharmaceutically acceptable salt thereof.

Embodiment 30

The pharmaceutical composition of embodiment 29, wherein R² is —CX^(2.1)₃, —(CH₂)_(n2)CX^(2.1) ₃ or C₂-C₄ haloalkyl.

Embodiment 31

The pharmaceutical composition of embodiment 29 or 30, wherein R³, R⁴and R⁵ are independently hydrogen, or substituted or unsubstituted C₁-C₄alkyl.

Embodiment 32

The pharmaceutical composition of embodiment 29, 30 or 31, wherein: R¹is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1), —CN, —NO₂,—C(O)R^(1D), —C(O)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂ or substituted orunsubstituted alkyl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃, —CHX⁶. 2,—CH₂X^(6.1), —CN, —NO₂, —C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂ or substituted or unsubstituted alkyl; R⁷ is hydrogen,—CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D),—C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂ or substituted orunsubstituted alkyl; R⁸ is hydrogen, —CX^(8.1) ₃, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; and R⁹ is hydrogen,halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —NO₂, —C(O)R^(9D),—C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl.

Embodiment 33

The pharmaceutical composition of embodiment 32, wherein R⁷ and R⁸ areindependently hydrogen.

Embodiment 34

The pharmaceutical composition of embodiment 29 or 30, wherein: R³ andR⁴ are independently methyl or ethyl; R⁵ are independently hydrogen; R⁶is hydrogen, halogen, —CX^(6.1) ₃, —CHX^(6.1) ₂, —CH₂X^(6.1), —CN,—C(O)R^(6D), —C(O)OR^(6D), —OCX^(6.1) ₃, —OCHX^(6.1) ₂ or substituted orunsubstituted alkyl; R⁷ is hydrogen, —CX^(7.1) ₃, —CHX^(7.1) ₂,—CH₂X^(7.1), —CN, —NO₂, —C(O)R^(7D), —C(O)OR^(7D), —OCX^(7.1) ₃, —OCHX⁷²or substituted or unsubstituted alkyl; R¹ is hydrogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —NO₂, —C(O)R^(8D), —C(O)OR^(8D),—OCX^(8.1) ₃, —OCHX^(8.1) ₂ or substituted or unsubstituted alkyl; andR⁹ is hydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—C(O)R^(9D), —C(O)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂ or substituted orunsubstituted alkyl; wherein R^(1D), R^(6D), R^(7D), R^(8D) and R^(9D)are independently hydrogen or methyl, and X^(1.1), X^(2.1), X^(6.1),X^(7.1), X^(8.1) and X^(9.1) are independently are —F.

Embodiment 35

The pharmaceutical composition of embodiment 34, wherein: R¹ ishydrogen, halogen, —NO₂, or —⁻COOH; R⁵ is hydrogen; R⁶ is hydrogen,halogen —NO₂, or —COOH; R⁷ and R⁸ are independently hydrogen; and R⁹ ishydrogen, halogen NO₂, or —COOH.

Embodiment 36

The pharmaceutical composition of embodiment 29 or 30, wherein R³ and R⁴are independently hydrogen, methyl or ethyl.

Embodiment 37

The pharmaceutical composition of embodiment 36, wherein R² is C₂-C₄alkyl substituted with at least one fluorine.

Embodiment 38

The pharmaceutical composition of embodiment 37, wherein R¹, R⁶, R⁷, R⁸and R⁹ are independently hydrogen.

Embodiment 39

The pharmaceutical composition of embodiment 29 or 30, wherein: R¹ andR⁶ are joined to form, together with the atoms to which they areattached, a substituted or unsubstituted pyrazolyl, oxazolyl, orthiazolyl; and R³ and R⁴ are independently hydrogen, methyl or ethyl.

Embodiment 40

The pharmaceutical composition of embodiment 39, wherein the compound isrepresented with Formula IB, IC, ID, or IE:

Embodiment 41

The pharmaceutical composition of embodiment 40, wherein R⁵, R⁷, R⁸ andR⁹ are independently hydrogen.

Embodiment 42

The pharmaceutical composition of embodiment 30, wherein R² is —CH(CF₃)₂or —CH₂(CF₂)₂H.

Embodiment 43

The pharmaceutical composition of embodiment 24, wherein the compound isselected from:

Embodiment 44

A pharmaceutical composition, comprising a pharmaceutically acceptableexcipient, and compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—; R² isC₂-C₄ haloalkyl; R³ is hydrogen; R is —CH₃ or —CH₂CH₃; and R¹, R⁶, R⁷,R⁸ and R⁹ are hydrogen.

Embodiment 45

The pharmaceutical composition of embodiment 44, wherein R² issubstituted with at least four fluorines.

Embodiment 46

A method of treating constipation in a subject in need thereof,comprising administering to the subject an effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—, —NH—or —S—; n1, n2, n6, n7, n8, and n9 are independently an integer from 0to 4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently1 or 2; R¹ is hydrogen, halogen, —CX^(1.13), —CHX^(1.1) ₂, —CH₂X^(1.1),—CN, —SO_(n1)R^(1B)R^(1C), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)CX^(2.1) ₃, —OR^(2A), substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl; R¹ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl, wherein R³ and R⁴ are optionally be joinedto form, together with the atoms to which they are attached, asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n)R^(6A), —SO₆NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(5B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D),—C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C),—NR^(7B)C(O)OR^(7C), —NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A),—SO_(v8)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m8),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX⁹³,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹ andR⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ are optionally joined to form,together with the atoms to which they are attached, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R^(1A), R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C),R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D),R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A),R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B),R^(9C) and R^(9D) are independently hydrogen, halogen, —CF₃—C₃, —CBr₃,—Cl₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO_(3H), —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH,—NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C),R^(3B), R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B),R^(7C), R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to thesame nitrogen atom may optionally be joined to form, together with theatoms to which they are attached, a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) ₁ areindependently —CF₁, —Br, —I or —F; with the proviso that when X is —O—;R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R¹ is substituted or unsubstituted C₁-C₃ alkyl, then R⁶ isnot —NO₂; and when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R¹ is substituted orunsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂.

Embodiment 47

The method of embodiment 46, further comprising administering to thesubject an anti-constipation agent.

Embodiment 48

The method of embodiment 46 or 47, wherein the compound is administeredorally.

Embodiment 49

The method of embodiment 46, 47 or 48, wherein the constipation isopioid-induced constipation, chronic idiopathic constipation orirritable bowel syndrome with constipation predominance.

Embodiment 50

A method of treating a dry eye disorder in a subject in need thereof,comprising administering to the subject an effective amount a compoundof Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—, —NH—or —S—; n1, n2, n6, n7, n8, and n9 are independently an integer from 0to 4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently1 or 2; R¹ is hydrogen, halogen, —CX^(1.13), —CHX^(1.1) ₂, —CH₂X^(1.1),—CN, —SO_(v1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl; R⁴ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl, wherein R³ and R⁴ are optionally be joinedto form, together with the atoms to which they are attached, asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6C)(O)R^(6D), —NR^(6C)(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R⁷, —C(O)OR^(7D), —C(O)NR^(7B)R^(7C),—OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7C)(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ is hydrogen, halogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v1)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(8B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C),—N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR⁹, —C(O)NR^(9B)R^(9C),—OR^(9A), —NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR^(9D),—NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹are optionally joined to form, together with the atoms to which they areattached, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1A), R^(1B), R^(1C), R^(1D),R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A),R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B),R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A), R^(5B), R^(5C),R^(5D), R^(9A), R^(9B), R^(9C) and R^(9D) are independently hydrogen,halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂,—OCHCl₂, —OCHBr₂, —OCHI₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;R^(1B), R^(1C), R^(2B), R^(2C)R^(3B), R^(3C), R^(4B), R^(4C), R^(5B),R^(5C), R^(6B), R^(6C), R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) andR^(9C) substituents bonded to the same nitrogen atom may optionally bejoined to form, together with the atoms to which they are attached, asubstituted or unsubstituted heterocycloalkyl or substituted orunsubstituted heteroaryl; and X^(1.1), X^(2.1), X^(6.1), X^(7.1),X^(8.1) and X^(9.1) are independently —Cl, —Br, —I or —F; with theproviso that when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R is substituted orunsubstituted C₁-C₃ alkyl, then R⁶ is not —NO₂; and when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is substituted or unsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂.

Embodiment 51

The method of embodiment 50, wherein the dry eye disorder is a lacrimalgland disorder.

Embodiment 52

The method of embodiment 50 or 51, further comprising administering tothe subject an anti-dry eye agent.

Embodiment 53

A method of increasing lacrimation in a subject in need thereof,comprising administering to the subject an effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:X is —O—, —NH— or —S—; n1, n2, n6, n7, n8, and n9 are independently aninteger from 0 to 4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are eachindependently 1 or 2; R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1)₂, —CH₂X^(1.1), —CN, —SO₁, R^(1A), —SO_(v1)NR^(1B)R^(1C),—NHNR^(1B)R^(1C), —ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C),—NHC(O)NR^(1B)R^(1C), —N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D),—C(O)OR^(1D), —C(O)NR^(1B)R^(1C), —OR^(1A), —ONR^(1B)SO₂R^(1A),—NR^(1B)C(O)R^(1D), —NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃,—OCHX^(1.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R² ishydrogen, —CX^(2.1) ₃, —CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl or haloalkyl; R³ is hydrogen,—C(O)R^(3D), —C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A),—C(O)NR^(3B)R^(3C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl; R⁴ ishydrogen, —C(O)R^(4D), —C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A),—C(O)NR^(4B)R^(4C), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, wherein R³and R⁴ may optionally be joined to form, together with the atoms towhich they are attached, a substituted or unsubstituted heterocycloalkylor substituted or unsubstituted heteroaryl, wherein R³ and R⁴ areoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6C)(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R^(7D), —C(O)OR^(7D),—C(O)NR^(7B)R^(7C), —OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C),—NR^(7B)C(O)OR^(7C), —NR^(7B)OR^(7C), —OCX^(7.1) ₃, —OCHX^(7.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁸ is hydrogen, halogen,—CX^(8.1) ₃, —CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A),—SO_(v8)NR^(8B)R^(8C), —NHNR^(8B)R^(8C), —ONR^(8B)R^(8C),—NHC(O)NHNR^(8B)R^(8C), —NHC(O)NR^(8B)R^(8C), —N(O)_(m8),—NR^(8B)R^(8C), —C(O)R^(8D), —C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A),—NR^(8B)SO₂R^(8A), —NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D),—NR^(8B)OR^(8D), —OCX^(8.1) ₃, —OCHX^(8.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁹ is hydrogen, halogen, —CX^(9.1) ₃,—CHX^(9.1) ₂, —CH₂X^(9.1), —CN, —SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C),—NHNR^(9B)R^(9C), —ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C),—NHC(O)NR^(9B)R^(9C), —N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D),—C(O)OR^(9D), —C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A),—NR^(9B)C(O)R^(9D), —NR^(9B)C(O)OR, —NR^(9B)OR^(9D), —OCX^(9.1) ₃,—OCHX^(9.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹ andR⁶, R⁶ and R⁷, R¹ and R⁹, or R⁸, and R⁹ are optionally joined to form,together with the atoms to which they are attached, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R^(1A), R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C),R^(2D), R^(3A), R^(3B), R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D),R^(5A), R^(5B), R^(5C), R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A),R^(7B), R^(7C), R^(7D), R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B),R^(9C) and R^(9D) are independently hydrogen, halogen, —CF₃—CCl₃, —CBr₃,—CI₃, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH,—NHOH, —OCF₃, —OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R^(1B), R^(1C), R^(1D), R^(2C),R^(3B), R^(3C), R^(3D), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C),R^(7B), R^(7C), R^(8B), R^(8C), R^(9B) and R^(9C)-substituents bonded tothe same nitrogen atom may optionally be joined to form, together withthe atoms to which they are attached, a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F; with the proviso that when X is —O—;R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R¹ is substituted or unsubstituted C₁-C₃ alkyl, then R⁶ isnot —NO₂; and when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R¹ is substituted orunsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂.

Embodiment 54

A method of activating a Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR), comprising contacting the CFTR with an effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—, —NH—or —S—; n, n2, n6, n7, n8, and n9 are independently an integer from 0 to4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently 1or 2; R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1),—CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl; R⁴ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl, wherein R³ and R⁴ are optionally be joinedto form, together with the atoms to which they are attached, asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(1B)R^(1C), —C(O)OR′, —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)(O)R^(6D), —NR^(6B)(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R⁷, —C(O)OR^(7D), —C(O)NR^(7B)R^(7C),—OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7C)(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁸ is hydrogen, halogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(8D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(5D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(8B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C),—N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D),—C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D),—NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ andR⁹, or R⁸, and R⁹ are optionally joined to form, together with the atomsto which they are attached, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(1A),R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B),R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C),R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D),R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) areindependently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B),R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C),R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to the samenitrogen atom may optionally be joined to form, together with the atomsto which they are attached, a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F; with the proviso that when X is —O—;R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R⁴ is substituted or unsubstituted C₁-C₃ alkyl, then R⁶ isnot —NO₂; and when X is —O—; R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1;X^(2.1) is fluorine; R³ is hydrogen; and R is substituted orunsubstituted C₁-C₃ alkyl, then R⁹ is not —NO₂.

Embodiment 55

A method of treating a cholestatic liver disease in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—, —NH—or —S—; n1, n2, n6, n7, n8, and n9 are independently an integer from 0to 4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently1 or 2; R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1),—CN, —SO_(n1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl; R⁴ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl, wherein R³ and R⁴ are optionally be joinedto form, together with the atoms to which they are attached, asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B)R^(6C),—NHNR^(6B)R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—(C)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R⁷, —C(O)OR^(7D), —C(O)NR^(7B)R^(7C),—OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ is hydrogen, halogen, —CX^(8.1) ₃,—CHX^(8.1) ₂, —CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v1)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(1D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n9)R^(9A), —SO_(v9)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C),—N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D),—C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D),—NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ andR⁹, or R⁸, and R⁹ are optionally joined to form, together with the atomsto which they are attached, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(1A),R^(1B), R^(1C), R^(1D), R^(2A), R^(2C)R^(2D), R^(3A), R^(3B), R^(3C),R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C), R^(5D),R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D), R^(8A),R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) areindependently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B),R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C)R^(7B), R^(7C),R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to the samenitrogen atom may optionally be joined to form, together with the atomsto which they are attached, a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F; with the proviso that when X is —O—;R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R is —CH₃, then R⁶ is not —NO₂; and when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ is hydrogen; andR is —CH₃, then R⁹ is not —NO₂.

Embodiment 56

A method of treating a pulmonary disease or disorder in a subject inneed thereof, the method comprising administrating to the subject aneffective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—, —NH—or —S—; n, n2, n6, n7, n8, and n9 are independently an integer from 0 to4; m1, m6, m7, m8, m9, v1, v6, v7, v8, and v9 are each independently 1or 2; R¹ is hydrogen, halogen, —CX^(1.1) ₃, —CHX^(1.1) ₂, —CH₂X^(1.1),—CN, —SO_(v1)R^(1A), —SO_(v1)NR^(1B)R^(1C), —NHNR^(1B)R^(1C),—ONR^(1B)R^(1C), —NHC(O)NHNR^(1B)R^(1C), —NHC(O)NR^(1B)R^(1C),—N(O)_(m1), —NR^(1B)R^(1C), —C(O)R^(1D), —C(O)OR^(1D),—C(O)NR^(1B)R^(1C), —OR^(1A), —NR^(1B)SO₂R^(1A), —NR^(1B)C(O)R^(1D),—NR^(1B)C(O)OR^(1D), —NR^(1B)OR^(1D), —OCX^(1.1) ₃, —OCHX^(1.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R² is hydrogen, —CX^(2.1) ₃,—CHX^(2.1) ₂, —(CH₂)_(n2)CX^(2.1) ₃, —OR^(2A), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or haloalkyl; R³ is hydrogen, —C(O)R^(3D),—C(O)NHNR^(3B)R^(3C), —C(O)OR^(3D), —SO₂R^(3A), —C(O)NR^(3B)R^(3C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl; R⁴ is hydrogen, —C(O)R^(4D),—C(O)NHNR^(4B)R^(4C), —C(O)OR^(4D), —SO₂R^(4A), —C(O)NR^(4B)R^(4C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl, wherein R³ and R⁴ are optionally be joinedto form, together with the atoms to which they are attached, asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁵ is hydrogen, —C(O)R^(5D),—C(O)NHNR^(5B)R^(5C), —C(O)OR^(5D), —SO₂R^(5A), —C(O)NR^(5B)R^(5C),substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, wherein R³ and R⁴ mayoptionally be joined to form, together with the atoms to which they areattached, a substituted or unsubstituted heterocycloalkyl or substitutedor unsubstituted heteroaryl; R⁶ is hydrogen, halogen, —CX^(6.1) ₃,—CHX^(6.1) ₂, —CH₂X^(6.1), —CN, —SO_(n6)R^(6A), —SO_(v6)NR^(6B), R^(6C),—NHNR^(6B), R^(6C), —ONR^(6B)R^(6C), —NHC(O)NHNR^(6B)R^(6C),—NHC(O)NR^(6B)R^(6C), —N(O)_(m6), —NR^(6B)R^(6C), —C(O)R^(6D),—C(O)OR^(6D), —C(O)NR^(6B)R^(6C), —OR^(6A), —NR^(6B)SO₂R^(6A),—NR^(6B)C(O)R^(6D), —NR^(6B)C(O)OR^(6D), —NR^(6B)OR^(6D), —OCX^(6.1) ₃,—OCHX^(6.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁷ ishydrogen, halogen, —CX^(7.1) ₃, —CHX^(7.1) ₂, —CH₂X^(7.1), —CN,—SO_(n7)R^(7A), —SO_(v7)NR^(7B)R^(7C), —NHNR^(7B)R^(7C),—ONR^(7B)R^(7C), —NHC(O)NHNR^(7B)R^(7C), —NHC(O)NR^(7B)R^(7C),—N(O)_(m7), —NR^(7B)R^(7C), —C(O)R, —C(O)OR^(7D), —C(O)NR^(7B)R^(7C),—OR^(7A), —NR^(7B)SO₂R^(7A), —NR^(7A)C(O)R^(7C), —NR^(7B)C(O)OR^(7D),—NR^(7B)OR^(7D), —OCX^(7.1) ₃, —OCHX^(7.1) ₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R¹ is hydrogen, halogen, —CX¹³, —CHX^(8.1) ₂,—CH₂X^(8.1), —CN, —SO_(n8)R^(8A), —SO_(v8)NR^(8B)R^(8C),—NHNR^(8B)R^(8C), —ONR^(8B)R^(8C), —NHC(O)NHNR^(8B)R^(8C),—NHC(O)NR^(8B)R^(8C), —N(O)_(m8), —NR^(8B)R^(8C), —C(O)R^(8D),—C(O)OR^(1D), —C(O)NR^(8B)R^(8C), —OR^(8A), —NR^(8B)SO₂R^(8A),—NR^(8B)C(O)R^(8D), —NR^(8B)C(O)OR^(8D), —NR^(8B)OR^(8D), —OCX^(8.1) ₃,—OCHX^(8.1) ₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R⁹ ishydrogen, halogen, —CX^(9.1) ₃, —CHX^(9.1) ₂, —CH₂X^(9.1), —CN,—SO_(n1)R^(9A), —SO_(v1)NR^(9B)R^(9C), —NHNR^(9B)R^(9C),—ONR^(9B)R^(9C), —NHC(O)NHNR^(9B)R^(9C), —NHC(O)NR^(9B)R^(9C),—N(O)_(m9), —NR^(9B)R^(9C), —C(O)R^(9D), —C(O)OR^(9D),—C(O)NR^(9B)R^(9C), —OR^(9A), —NR^(9B)SO₂R^(9A), —NR^(9B)C(O)R^(9D),—NR^(9B)C(O)OR^(9D), —NR^(9B)OR^(9D), —OCX^(9.1) ₃, —OCHX^(9.1) ₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R¹ and R⁶, R⁶ and R⁷, R¹ andR⁹, or R⁸, and R⁹ are optionally joined to form, together with the atomsto which they are attached, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R^(1A),R^(1B), R^(1C), R^(1D), R^(2A), R^(2B), R^(2C), R^(2D), R^(3A), R^(3B),R^(3C), R^(3D), R^(4A), R^(4B), R^(4C), R^(4D), R^(5A), R^(5B), R^(5C),R^(5D), R^(6A), R^(6B), R^(6C), R^(6D), R^(7A), R^(7B), R^(7C), R^(7D),R^(8A), R^(8B), R^(8C), R^(8D), R^(9A), R^(9B), R^(9C) and R^(9D) areindependently hydrogen, halogen, —CF₃, —CCl₃, —CBr₃, —CI₃, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)—OH, —NHOH, —OCF₃,—OCCl₃, —OCBr₃, —OCI₃, —OCHF₂, —OCHCl₂, —OCHBr₂, —OCHI₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R^(1B), R^(1C), R^(2B), R^(2C), R^(3B),R^(3C), R^(4B), R^(4C), R^(5B), R^(5C), R^(6B), R^(6C), R^(7B), R^(7C),R^(8B), R^(8C), R^(9B) and R^(9C) substituents bonded to the samenitrogen atom may optionally be joined to form, together with the atomsto which they are attached, a substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heteroaryl; andX^(1.1), X^(2.1), X^(6.1), X^(7.1), X^(8.1) and X^(9.1) areindependently —Cl, —Br, —I or —F; with the proviso that when X is —O—;R² is —(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X^(2.1) is fluorine; R³ ishydrogen; and R is —CH₃, then R⁶ is not —NO₂; and when X is —O—; R² is—(CH₂)_(n2)CX^(2.1) ₃; n2 is 1; X²¹ is fluorine; R³ is hydrogen; and Ris —CH₃, then R⁹ is not —NO₂.

Embodiment 57

The method of embodiment 56, wherein the pulmonary disease or disorderis chronic obstructive pulmonary disease, bronchitis, asthma, andcigarette smoke-induced lung dysfunction.

Embodiment 58

A method of treating constipation, comprising administering to a.subject in need thereof a therapeutically effective amount a compound inany of embodiments 1 to 23.

Embodiment 59

The method of embodiment 58, further comprising administering to thesubject an anti-constipation agent.

Embodiment 60

The method of embodiment 58 or 59, wherein the compound is administeredorally.

Embodiment 61

The method of embodiment 58, 59 or 60, wherein the constipation isopioid-induced constipation, chronic idiopathic constipation orirritable bowel syndrome with constipation predominance.

Embodiment 62

A method of treating a dry eye disorder, comprising administering to asubject in need thereof a therapeutically effective amount of a compoundin any of embodiments 1 to 23.

Embodiment 63

The method of embodiment 62, wherein the dry eye disorder is a lacrimalgland disorder.

Embodiment 64

The method of embodiment 62 or 63, further comprising administering tothe subject an anti-dry eye agent.

Embodiment 65

A method of increasing lacrimation, comprising administering to asubject in need thereof a compound in any of embodiments 1 to 23.

Embodiment 66

A method of activating Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR), comprising contacting CFTR with a compound in any ofembodiments 1 to 23.

Embodiment 67

A method of treating a cholestatic liver disease in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a compound in any of embodiments 1 to 23.

Embodiment 68

A method of treating a pulmonary disease or disorder in a subject inneed thereof, the method comprising administrating to the subject aneffective amount of a compound in any of embodiments 1 to 23.

Embodiment 69

The method of embodiment 68, wherein the pulmonary disease or disorderis chronic obstructive pulmonary disease, bronchitis, asthma, andcigarette smoke-induced lung dysfunction.

V. Examples

Constipation

1. Example 1

A cell-based high-throughput screen was done for 120,000 drug-like,synthetic small molecules. Active compounds were characterized formechanism of action and one lead compound was tested in aloperamide-induced constipation model in mice.

Several classes of novel CFTR activators were identified, one of which,the phenylquinoxalinone CFTR_(act)-J027, fully activated CFTR chlorideconductance with EC₅₀˜200 nM, without causing elevation of cytoplasmiccAMP. Orally administered CFTR_(act)-J027 normalized stool output andwater content in a loperamide-induced mouse model of constipation withED₅₀˜0.5 mg/kg; CFTR_(act)-J027 was without effect in cystic fibrosismice lacking functional CFTR. Short-circuit current, fluid secretion andmotility measurements in mouse intestine indicated a pro-secretoryaction of CFTR_(act)-J027 without direct stimulation of intestinalmotility. Oral administration of 10 mg/kg CFTR_(act)-J027 showed minimalbioavailability, rapid hepatic metabolism and blood levels <200 nM, andwithout apparent toxicity after chronic administration.

CFTR_(act)-J027 or alternative small-molecule CFTR-targeted activatorsmay be efficacious for the treatment of constipation.

High-throughput screening was done using a diverse collection of 120,000drug-like synthetic compounds obtained from ChemDiv Inc. (San Diego,Calif., USA) and Asinex (Winston-Salem, N.C., USA). Forstructure-activity analysis, 600 commercially available analogs (ChemDivInc.) of active compounds identified in the primary screen were tested.Other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA)unless indicated otherwise.

CFTR_(act)-J027 synthesis

To a solution of o-phenylenediamine (1 g, 9.24 mmol) in DMF (30 mL) wasadded potassium carbonate (2.5 g, 18.4 mmol) and benzyl bromide (0.73mL, 6.2 mmol) then stirred overnight at ambient temperature. Thereaction mixture was diluted with CH₂Cl₂, washed with water, dried overMgSO₄ and concentrated under reduced pressure. The residue was purifiedby flash chromatography to give the intermediateN¹-benzylbenzene-1,2-diamine as a brown liquid. ¹H NMR (300 MHz, CDCl₃):δ 7.45-7.31 (m, 5H), 6.86-6.69 (m, 4H), 4.35 (s, 2H), 3.50 (br, 3H); MS:m/z 199 (M+H). Then, a solution of the intermediate (400 mg, 2 mmol) and5-nitroisatin (380 mg, 2 mmol) in acetic acid (5 mL) was refluxed for 2h. The reaction mixture was cooled to room temperature and solventremoved under reduced pressure. The residue was dissolved with methanoland acetic acid was added to crystallize3-(2-amino-5-nitrophenyl)-1-benzylquinoxalin-2(1H)-one (CFTR_(act)-J027)as a yellow powder with >99% purity. ¹H NMR (300 MHz, DMSO-d6): δ 9.15(d, 1H, J=2.8 Hz), 8.07 (dd, 1H, J=2.7, 9.2 Hz), 7.97 (dd, 1H, J=1.2,7.9 Hz), 7.82 (brs, 2H), 7.60-7.27 (m, 7H), 6.92 (d, 1H, J=9.2 Hz), 5.59(brs, 2H); ¹³C NMR (75 MHz, DMSO-d6): δ 155.0, 154.6, 153.3, 136.3,135.3, 132.8, 132.2, 131.0, 130.0, 129.5, 129.1, 127.7, 127.3, 126.8,124.1, 116.1, 115.9, 115.4, 45.9; MS: m/z 373 (M+H).

Cell Culture

Fischer Rat Thyroid (FRT) cells stably co-expressing human wild-typeCFTR and the halide-sensitive yellow fluorescent protein (YFP)-H148Qwere generated as previously described [12]. Cells were cultured onplastic in Coon's-modified Ham's F12 medium supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/mlstreptomycin. For high-throughput screening, cells were plated in black96-well microplates (Corning-Costar Corp., Corning, N.Y., USA) at adensity of 20,000 cells per well. Screening was done 24-48 hours afterplating.

High-Throughput Screening

Screening was carried out using a Beckman Coulter integrated systemequipped with a liquid handling system and two FLUOstar fluorescenceplate readers (BMG Labtechnologies, Durham, N.C., USA), each equippedwith dual syringe pumps and 500±10 nm excitation and 535±15 nm emissionfilters (details in ref. 12). CFTR- and YFP-expressing FRT cells weregrown at 37° C./5% CO₂ for 24-48 hours after plating. At the time ofassay, cells were washed three times with phosphate-buffered saline(PBS) and then incubated for 10 min with 60 μl of PBS containing testcompounds (at 10 μM) and a low concentration of forskolin (125 nM). Eachwell was assayed individually for I⁻ influx in a plate reader byrecording fluorescence continuously (200 ms per point) for 2 s(baseline) and then for 12 s after rapid (<1 s) addition of 165 μL ofPBS in which 137 mM Cl⁻ was replaced by I⁻. The initiate rate of I⁻influx was computed by determined using exponential regression. Allcompound plates contained negative controls (DMSO vehicle) and positivecontrols (20 μM forskolin).

Short-Circuit Current Measurement

Short-circuit current was measured in FRT cells stably expressingwild-type human CFTR cultured on porous filters as described [12]. Thebasolateral solution contained 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1mM CaCl₂, 0.5 mM MgCl₂, 10 mM glucose, and 10 mM Na-HEPES (pH 7.3, 37°C.). In the apical solution 65 mM NaCl was replaced by Na gluconate, andCaCl₂ was increased to 2 mM, and the basolateral membrane waspermeabilized with 250 μg/ml amphotericin B. Short-circuit current wasmeasured in freshly harvested adult mouse colon at 37° C. usingsymmetrical Krebs-bicarbonate buffer.

cAMP Assay

Intracellular cAMP activity was measured using a GloSensor luminescenceassay (Promega Corp., Madison, Wis., USA). FRT null cells were stablytransfected with the pGloSensor cAMP plasmid and plated onto white96-well microplates and grown to confluence. Cells were washed threetimes with PBS and incubated with 5 μM CFTR_(act)-J027 for 10 min in theabsence and presence of 100 nM forskolin. cAMP was assayed according tothe manufacturer's instructions.

Phaarmacoklnecs

All animal experiments were approved by UCSF Institutional Animal Careand Use Committee. Female CD1 mice were treated with 10 mg/kgCFTR_(act)-J027 (saline containing 5% DMSO and 10% Kolliphor HS 15)either intraperitoneally (ip) or orally. Blood was collected at 15, 30,60, 150, 240 and 360 min after treatment by orbital puncture andcentrifuged at 5000 rpm for 15 min to separate plasma. Plasma samples(60 μL) were mixed with 300 μL acetonitrile and centrifuged at 13000 rpmfor 20 min, and 90 μL of the supernatant was used for LC/MS. The solventsystem consisted of a linear gradient from 5 to 95% acetonitrile over 16min (0.2 ml/min flow). Mass spectra was acquired on a mass spectrometer(Waters 2695 and Micromass ZQ) using electrospray (+) ionization, massranging from 100 to 1500 Da, cone voltage 40 V. Calibration standardswere prepared in plasma from untreated mice to which known amounts ofCFTR_(act)-J027 were added.

In Vita Metabolic Stability

CFTR_(act)-J027 (5 μM) was incubated for specified times at 37° C. withmouse liver microsomes (1 mg protein/ml; Sigma-Aldrich) in potassiumphosphate buffer (100 mM) containing 1 mM NADPH, as described [13]. Themixture was then chilled on ice, and 0.5 ml of ice-cold ethyl acetatewas added. Samples were centrifuged for 15 min at 3000 rpm, thesupernatant evaporated to dryness, and the residue was dissolved in 100μL mobile phase (acetonitrile:water, 3:1) for LC/MS and assayed asdescribed above.

Murine Model of Constipation

Female CD1 mice (age 8-10 weeks) were administered loperamide (0.3mg/kg, ip, Sigma-Aldrich) to produce constipation. Various amounts ofCFTR_(act)-J027 (0.1, 0.3, 1, 3 and 10 mg/kg) were given at the sametime (for ip administration) or 1 h before (for oral administration)loperamide. Control mice were treated with vehicle only. Some mice weretreated orally with lubiprostone (0.5 mg/kg, Sigma-Aldrich) orlinaclotide (0.5 mg/kg, Toronto Research Chemicals Inc., Toronto,Ontario, Canada). After loperamide injection, mice were placedindividually in metabolic cages with food and water provided ad libitum.Stool samples were collected for 3 h, and total stool weight and numberof fecal pellets were quantified. To measure stool water content stoolsamples were dried at 80° C. for 24 h and water content was calculatedas [wet weight-dry weight]/wet weight. Similar studies were done incystic fibrosis (CF) mice (ΔF508 homozygous) lacking functional CFTR.Some studies were done using the chemically similar but inactive analogof CFTR_(act)-J027,3-(2-amino-5-nitrophenyl)-1-(methyl)-2(1H)-quinoxalinone.

In Vivo Intestinal Transit and Ex Vivo Intestinal Contractility

Whole-gut transit time was determined using an orally administeredmarker (200 μL, 5% Evans Blue, 5% gum Arabic) and measuring the time ofits appearance in stool. Mice were administered loperamide andCFTR_(act)-J027 (10 mg/kg) or vehicle intraperitoneally at zero time.For ex vivo contractility measurements, mice were euthanized by avertinoverdose (200 mg/kg, 2,2,2-tribromethanol, Sigma-Aldrich) and ileum andcolon segments of ˜2 cm length were isolated and washed withKrebs-Henseleit buffer. The ends of the intestinal segments were tied,connected to a force transducer (Biopac Systems, Goleta, Calif., USA)and tissues were transferred to an organ chamber (Biopac Systems)containing Krebs-Henseleit buffer at 37° C. aerated with 95% O₂, 5% CO₂.Ileum and colon were stabilized for 60 min with resting tensions of 0.5and 0.2 g respectively, and solutions were changed every 15 min. Effectsof CFTR_(act)-J027 on baseline and loperamide-suppressed isometricintestinal contractions were recorded.

In Vivo Intestinal Secretion and Absorption

Mice (wildtype or CF) were given access to 5% dextrose water but notsolid food for 24 h before experiments. Mice were anesthetized withisoflurane and body temperature was maintained during surgery at 36-38°C. using a heating pad. A small abdominal incision was made to exposethe small intestine, and closed mid-jejunal loops (length 2-3 cm) wereisolated by sutures. Loops were injected with 100 μL vehicle alone or100 μg CFTR_(act)-J027 in vehicle. The abdominal incision was closedwith sutures, and mice were allowed to recover from anesthesia.Intestinal loops were removed at 90 min and loop length and weight weremeasured to quantify fluid secretion. Intestinal absorption was measuredin CF mice (to prevent secretion) as described above, except that theloops were removed at 0 or 30 min. Absorption was calculated as 1-(loopweight at 0 min−loop weight at 30 min)/loop weight at 0 min.

Chronic Administration and Toxicity Studies

Mice were administered 10 mg/kg CFTR_(act)-J027 or vehicle orally once aday for 7 d. One hour after the final dose mice were treated withloperamide (0.3 mg/kg, ip) and stool was collected for 3 h. In vivotoxicity was assessed in these mice by measuring lung wet/dry weightratio, complete blood count (HEMAVET 950FS, Drew Scientific Inc.,Florida, USA) and serum chemistry (Idexx Laboratories Inc., Sacramento,Calif., USA) 4 h after the last CFTR_(act)-J027 dose. In vitrocytotoxicity was measured in FRT cells incubated with 25 μMCFTR_(act)-J027 for 8 and 24 h. Cytotoxicity was measured by Alamar Blueassay according to the manufacturer's instructions (Invitrogen,Carlsbad, Calif., USA).

Statistical Analysis

Experiments with two groups were analyzed with Student's t-test, whenthere are 3 groups or more analysis was made with one-way analysis ofvariance and post-hoc Newman-Keuls multiple comparisons test. P<0.05 wastaken as statistically significant.

2. Example 2

Identification and In Vitro Characterization of Small-Molecule CFTRActivators

The goal was to identify a potent, CFTR-targeted activator withpro-secretory activity in intestine in order test its efficacy in amouse model of constipation. FIG. 1A summarizes the project strategy.The compounds evaluated here included small molecules identified inprior CFTR activator/potentiator screens [14] and from a new screen ofsynthetic small molecules not tested previously. The most activecompounds emerging from the screen, along with commercially availablechemical analogs, were prioritized based on an initial mechanism ofaction study (assay of cAMP elevation), in vitro toxicity, pro-secretoryaction in mouse intestine, and efficacy in a mouse model ofconstipation. FIG. 1B shows the cell-based plate reader screening methodin which the initial rate of iodide influx was measured in FRT cellsstably expressing human wildtype CFTR and a YFP fluorescent halidesensor following extracellular iodide addition. A CFTR activatorincreases the initial slope of the fluorescence quenching curve.

FIG. 1C shows chemical structures of six classes of CFTR candidateactivators identified from the screens. Based on the criteria listedabove, we focused further studies on CFTR_(act)-J027, a3-phenyl-quinoxalinone with drug-like properties. CFTR_(act)-J027 wassynthesized in pure crystalline form in two steps (FIG. 1D).

Short-circuit current measurements in CFTR-expressing FRT cells showedthat CFTR_(act)-J027 fully activated CFTR (FIG. 2A), as the cAMP agonistforskolin produced no further increase in current, with an EC₅₀˜200 nM(FIG. 2B). Interestingly, CFTR_(act)-J027 was only a weak potentiator ofΔF508-CFTR, as studied in FRT cells expressing ΔF508-CFTR afterovernight incubation with a corrector (FIG. 2C). Cl⁻ secretion infreshly isolated mouse colon showed a concentration-dependent increasein short-circuit current with EC₅₀˜300 nM (FIG. 2D). The increase incurrent at high CFTR_(act)-J027 was further increased by forskolin,which may be a consequence of activation of a basolateral membranecAMP-sensitive K⁺ channel that increases the driving force for apicalmembrane Cl⁻ secretion. The increase in current was fully inhibited by aCFTR-selective inhibitor. FIG. 2E shows that CFTR_(act)-J027 does notelevate cellular cAMP when added alone, and does not further increasecAMP when added together with forskolin, suggesting that CFTR activationinvolves a direct interaction mechanism rather than indirect actionthrough cAMP elevation.

CFTR_(act)-J027 Normalizes Stool Output in a Mouse Model of Constipation

CFTR_(act)-J027 was studied in the well-established loperamide-inducedmouse model of constipation in which stool weight, pellet number andwater content were measured over 3 h following intraperitonealloperamide administration (FIG. 3A). Intraperitoneal administration ofCFTR_(act)-J027 at 10 mg/kg normalized each of the stool parameters.CFTR_(act)-J027 did not affect stool output or water content in control(non-loperamide-treated) mice. Importantly, CFTR_(act)-J027 was withouteffect in cystic fibrosis mice lacking functional CFTR (FIG. 3B), norwas an inactive chemical analog of CFTR_(act)-J027 effective in wildtypemice (FIG. 3C). These results support a CFTR-selective action ofCFTR_(act)-J027. Dose-response studies in mice showed an ED₅₀ of 2 mg/kgin the loperamide model by ip administration of CFTR_(act)-J027 (FIG.3D).

Oral administration of 10 mg/kg CFTR_(act)-J027 1 h prior to loperamideadministration was also effective in normalizing stool output and watercontent in loperamide-treated mice, with no effect in control mice (FIG.4A). The ED₅₀ for oral administration was 0.5 mg/kg, substantially lowerthan that for ip administration (FIG. 4B). In parallel studies, oraladministration of the approved drugs lubiprostone or linaclotide at250-500 fold greater mg/kg doses than given to humans for treatment ofconstipation, were less effective in normalizing stool output, producing50% and 35% of the maximal CFTR_(act)-J027 response, respectively (FIG.4C).

CFTR_(act)-J027 Actions on Intestinal Transit, Motility and FluidTransport

CFTR_(act)-J027 action on intestinal transit and motility was measuredin vivo and in isolated intestinal strips, respectively. Whole-guttransit time, as measured by appearance of a marker in the stool afterbolus oral gavage at the time of ip loperamide and CFTR_(act)-J027administration, was normalized by CFTR_(act)-J027 (FIG. 5A, left panel).CFTR_(act)-J027 had no effect on whole-gut transit time in cysticfibrosis mice (right panel). In vitro measurements of intestinalcontraction showed no effect of CFTR_(act)-J027 added alone or in thepresence of 10 μM loperamide in isolated mouse ileum and colon strips(FIG. 5B). CFTR_(act)-J027 may thus increase intestinal transit in vivoby stimulating motility by secretion-induced stretch of the gut wall,without direct effect on intestinal smooth muscle.

To directly investigate the effects of CFTR_(act)-J027 on intestinalfluid secretion and absorption, an in vivo closed-intestinal loop modelwas used. CFTR_(act)-J027 was injected into closed, mid-jejunal loopsand fluid accumulation was measured at 90 min. CFTR_(act)-J027 produceda 140% increase in loop weight/length ratio, indicating fluid secretioninto the intestinal lumen in wild-type mice (FIG. 5C, upper panel), butwas without effect in cystic fibrosis mice (lower panel), supporting aCFTR-selective mechanism of action. A closed-loop model was also used tostudy CFTR_(act)-J027 action on intestinal fluid absorption. Fluidwithout or with CFTR_(act)-J027 was injected into closed, mid-jejunalloops of cystic fibrosis mice (to avoid confounding fluid secretion) andfluid absorption was measured at 30 min. CFTR_(act)-J027 did not affectintestinal fluid absorption (FIG. 5D).

CFTR_(act)-J027 Pharmacology and Toxicity in Mice

The in vitro metabolic stability of CFTR_(act)-J027 was measured byincubation with mouse liver microsomes in the presence of NADPH.CFTR_(act)-J027 was rapidly metabolized with ˜21 min eliminationhalf-life, with only 7% of the original compound remaining at 60 min(FIG. 6A).

Pharmacokinetics was measured in mice following bolus intraperitoneal ororal administration of 10 mg/kg CFTR_(act)-J027. Following ipadministration serum CFTR_(act)-J027 concentration decreased with anelimination half-life of ˜16 min, and was undetectable at 150 min (FIG.6B). Following oral administration serum CFTR_(act)-J027 concentrationreached 180 nM at 30 min and was undetectable at other time points (FIG.6B).

Preliminary toxicological studies of CFTR_(act)-J027 were done in cellcultures and mice. CFTR_(act)-J027, at a concentration of 20 μM near itssolubility limit, did not show cytotoxicity as measured by the AlamarBlue assay (FIG. 6C). In the 7-day treated mice, CFTR_(act)-J027 did notaffect the major serum chemistry and blood parameters (Table 1), nor didit change body weight or produce airway/lung fluid accumulation (FIG.6D).

Last, to determine whether chronically administered CFTR_(act)-J027retained efficacy, mice were treated orally for 7 days with 10 mg/kgCFTR_(act)-J027 or vehicle, and loperamide was given 1 h after the finaldose. FIG. 6E shows that chronically administered CFTR_(act)-J027remained effective in normalizing stool output and water contentfollowing loperamide.

TABLE 1 Complete blood count and serum chemistries of mice treated for 7days with 10 mg/kg CFTR_(act)-J027 or vehicle orally once per day (mean± S.E., 5 mice per group). Student's t-test. Vehicle CFTR_(act)-J027 Pvalue Hemoglobin (g/dL) 13.3 ± 0.2  12.8 ± 0.3 >0.05 Leukocytes (10³/μL)1.9 ± 0.3  1.9 ± 0.5 >0.05 Thrombocytes (10³/μL) 790 ± 109 900 ±48 >0.05 Total protein (g/dL) 4.7 ± 0.2  5.2 ± 0.1 >0.05 Albumin (g/dL)2.6 ± 0.1  2.9 ± 0.03 >0.05 Globulin (g/dL) 2.1 ± 0.1  2.2 ± 0.1 >0.05ALT (U/L) 52 ± 16 44 ± 6 >0.05 AST (U/L) 131 ± 17  105 ± 11 >0.05 ALP(U/L)  47 ± 8.5  53 ± 2.5 >0.05 Total bilirubin (mg/dL) 0.1 ± 0  0.1 ±0  >0.05 Glucose (mg/dL) 156 ± 22  164 ± 6  >0.05 Cholesterol (mg/dL)121 ± 14  121 ± 6  >0.05 CK (U/L) 344 ± 85  312 ± 62 >0.05 Sodium(mmol/L) 149 ± 2.3   151 ± 0.7 >0.05 Potassium (mmol/L) 5.0 ± 0.1  4.4 ±0.1 >0.05 Chloride (mmol/L) 113 ± 1  115 ± 1  >0.05 Calcium (mg/dL) 8.5± 0.2  8.5 ± 0.04 >0.05 Phosphorus (mg/dL) 6.6 ± 0.9  6.8 ± 0.3 >0.05BUN (mg/dL) 15.3 ± 3   18.4 ± 1.2 >0.05 Creatinine (mg/dL) 0.2 ± 0  0.2± 0  >0.05 Bicarbonate (mmol/L) 15.3 ± 1.6   16 ± 1.7 >0.05Dry Eye

3. Example 3

Mice

Wild-type (WT) and CF (homozygous ΔF508-CFTR mutant) mice in a CD1genetic background were bred at the University of California SanFrancisco (UCSF) Animal Facility. Mice aged 8 to 12 weeks (25 to 35 g)were used. Female BALB/c mice (7-8 weeks old) were purchased from theHarlan Laboratory (Livermore, Calif., USA). Animal protocols wereapproved by the UCSF Institutional Animal Care and Use Committee andwere in compliance with the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Short-Circuit Current

Fischer rat thyroid (FRT) cells stably expressing wild-type human CFTRwere cultured on Snapwell inserts (Corning Costar, Corning N.Y., USA)for short-circuit current (I_(sc)) measurements. After 6-9 days inculture, when the transepithelial resistance was >1000 Ω/cm², theinserts were mounted in an Ussing chamber system (World PrecisionInstruments, Sarasota, Fla., USA). The basolateral solution contained130 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1 mM CaCl₂, 0.5 mM MgCl₂, 10 mMglucose, and 10 mM Na-HEPES (pH 7.3). In the apical bathing solution, 65mM NaCl was replaced by Na gluconate, and CaCl₂ was increased to 2 mM.Both solutions were bubbled with air and maintained at 37° C. Thebasolateral membrane was permeabilized with 250 μg/ml amphotericin B(26, 27). Hemichambers were connected to a DVC-1000 voltage clamp viaAg/AgCl electrodes and 3 M KCl agar bridges for I_(sc) recording.

cAMP and Cytotoxicity Assays

Intracellular cAMP activity was measured using a GloSensor luminescenceassay (Promega Corp., Madison, Wis., USA). FRT cells stably transfectedwith the pGloSensor cAMP plasmid (Promega Corp.) were cultured in white96-well microplates (Corning Costar) overnight. Cells were then washedthree times with PBS and incubated with 5 μM test compound for 10 min inthe absence and presence of 100 nM forskolin. To assay cytotoxicity, FRTcells were cultured overnight in black 96-well Costar microplate wellsand incubated with test compounds at up to 100 μM (the maximumsolubility in PBS) for 1 or 24 h. Cytotoxicity was measured by AlamarBlue assay according to the manufacturer's instructions (Invitrogen,Carlsbad, Calif., USA).

Ocular Surface Potential Difference Measurements

Open-circuit transepithelial PD were measured continuously inanesthetized mice in response to serial perfusions of differentsolutions over the ocular surface, as described (21). Mice wereanesthetized with Avertin (2,2,2-tribromoethanol, 125 mg/kgintraperitoneal, Sigma-Aldrich, St. Louis, Mo., USA), and coretemperature was maintained at 37° C. using a heating pad. Eyes wereoriented with the cornea and conjunctiva facing upward and exposed byretracting the eyelid with cross-action forceps. Solutions wereisosmolar (320±10 mOsM; compositions provided in ref. 21) and contained10μ□□ indomethacin to prevent CFTR activation by prostaglandins. Theocular surface was perfused at 6 mL/min through plastic tubing using amultireservoir gravity pinch-valve system (ALA Scientific, Westbury,N.Y., USA) and variable-flow peristaltic pump (medium flow model; FisherScientific, Fair Lawn, N.J., USA). A probe catheter was fixed 1 mm abovethe comea using a micropositioner and a suction cannula was positioned 3mm from the orbit. The measuring electrode was in contact to theperfusion catheter and connected to a high-impedance voltmeter(IsoMilivolt Meter; WPI). The reference electrode was grounded via awinged 21-gauge needle filled with isosmolar saline, and insertedsubcutaneously in the abdomen. Measuring and reference electrodesconsisted of Ag/AgCl with 3 M KCl agar bridges.

Tear Secretion

To measure unstimulated tear production, phenol red threads (Zone-Quick,Oasis Medical, Glendora, Calif., USA) were placed for 10 s in thelateral canthi of isofluorane-anesthetized mice using jewelers' forceps.Tear volume was measured as the length of thread wetting, as visualizedunder a dissecting microscope. Serial measurements were used to evaluatecompound pharmacodynamics after application of 2-μL drops of compoundformulations (50-100 μM compound in PBS containing 0.5% polysorbate and0.5% DMSO) comparing to vehicle.

Lissamine Green Staining

To assess corneal epithelial disruption, 5 μL of lissamine green (LG)dye (1%) was applied to the ocular surface of isofluorane-anesthetizedmice. Photographs of the eye were taken using a Nikon Digital cameraadapted to an Olympus Zoom Stereo Microscope (Olympus, Center Valley,Pa., USA). Each corneal quadrant was scored on a 3-point scale by oneblinded, trained observer, with the extent of staining in each quadrantclassified as: 0, no staining; 1, sporadic (involving <25% of the totalsurface) staining; grade 2, diffuse punctate staining (25-75%); andgrade 3, coalesced punctate staining (275%). The total grade is reportedas the sum of scores from all four quadrants, ranging from 0 to 12.

Pharmacokinetics and Issue Distribution

To determine the residence time of CFTR activators in the pre-ocularmouse tear film, compounds were recovered for liquid chromatography/massspectroscopy (LC/MS) following single-dose ophthalmic delivery. Threeeye washes (3 μL PBS each) were recovered from the lateral and medialcanthi with 5-μL microcapillary tubes (Drummond Scientific Co.,Broomhall, Pa., USA) after manual eyelid blinking (9). Pooled washeswere diluted with acetonitrile/water (1:1) containing 0.1% formic acidand analyzed by LC/MS using an Xterra MS C18 column (2.1 mm×100 mm,3.5-μm particle size) connected to a Waters 2695 HPLC solvent deliverysystem and a Waters Micromass ZQ mass spectrometer with positiveelectrospray ionization.

To study compound accumulation in systemic tissues, mouse blood, brain,kidney and liver were analyzed after 14 days of three-times dailytopical dosing (0.1 nmol, 2 μL, 50 μM). Blood samples were collectedfrom the left ventricle into K3 EDTA mini-tubes (Greiner, Kremsmunster,Austria) and centrifuged (28). The supernatant was extracted with anequal volume of ethyl acetate and the extract was dried with an airstream. Organs from treated and control mice were removed followingventricular perfusion with heparinized PBS (10 units/mL), weighed, mixedwith acetic acid and water (100 μL/g tissue), and homogenized (29).Ethyl acetate (10 mL/g tissue) was added, samples were vortexed andcentrifuged (3000 rpm for 15 min), and the ethyl acetate-containingsupernatant was evaporated. Residues obtained from organic extracts ofserum and organ homogenates were then reconstituted and analyzed byLC/MS as described above.

Mouse Model of Dry Eye Produced by Lacrimal Gland Excision

A lacrimal gland excision (LGE) model of aqueous-deficient dry eye wasadapted from a reported method (30). The extraorbital lacrimal gland wasexposed on each side of wild-type female BALB/c mice (7-8 weeks of age)by 3-mm linear skin incisions. Lacrimal ducts were cauterized and theentire gland was removed bilaterally, avoiding facial vessels andnerves. Incisions were each closed with a single interrupted 6-0 silksuture. Orbital lacrimal tissue remained functional. Eyes with reducedcorneal sensation (<5% of mice studied), as identified from neurotrophiccorneal ulcers within 1 day of LGE, were excluded. Mice were randomizedto receive either treatment (in both eyes) with CFTR_(act)-K089 (0.1nmol) or vehicle. Mice were treated three times daily (8 AM, 2 PM and 8PM) for 2 weeks starting on Day 1 after LGE. Tear secretion and LGstaining were performed immediately prior to, and one hour after theinitial dose on day 4, 10 and 14 after LGE.

Statistics

Data are expressed as the mean±standard error of the mean (SEM). Fordirect comparisons between two means, the two-sided Students' t-test wasused. For longitudinal measurements of tear secretion and LG scores inthe dry eye prevention study, a linear mixed effects regression wasused, adjusting for non-independence of measurements taken on the sameeye and on both eyes of the same animal. Analysis was conducted in Rv.3.2 for Mac (R Foundation for Statistical Computing, Vienna, Austria),using packages lme4 and robustlmm.

Characterization of Small-Molecule CFTR Activators

A cell-based functional high-throughput screen of 120,000 compounds at10 μM identified 20 chemical classes of small-molecule activators ofwild-type CFTR that produced >95% of maximal CFTR activation. The screenwas done in FRT epithelial cells co-expressing human wild-type CFTR anda cytoplasmic YFP halide sensor in 96-well format (26, 31, 32).Secondary screening involved I_(sc) measurement in CFTR-expressing FRTcells pretreated with submaximal forskolin (50 nM). Twenty-one compoundsfrom eight chemical classes produced large increases in I_(sc) at 1μ□(>75% of maximal current produced by 20 μM forskolin). A summary of EC₅₀and V_(max) values for each compound is provided in FIG. 7.

Structures of activators from the four most active chemical classes areshown in FIG. 2A, along with corresponding concentration-dependence datafrom I_(sc) measurements. Each compound fully activated CFTR, as a highconcentration of forskolin produced little further increase in I_(sc),and the increase in I_(sc) was fully inhibited by a CFTR inhibitor,CFTR_(inh)-172. EC₅₀ values ranged from 20-350 nM (FIG. 2B). VX-770showed relatively weak activity against wild-type CFTR (FIG. 2C).CFTR_(act)-K032 and CFTR_(act)-K089 showed incomplete CFTR activation(˜50% V_(max)).

Compounds that directly target CFTR without causing elevation ofcellular cAMP were sought to minimize potential off-target effects (FIG.2D). Compounds producing elevations in intracellular cAMP (from ClassesO, Q, and R), probably by phosphodiesterase inhibition, were excludedfrom further consideration. Nanomolar-potency compounds from Classes B,J and K, which did not increase cAMP, were selected for furthercharacterization in living mice.

CFTR Activators Increase Ocular Surface Chloride and Fluid Secretion InVive

An open-circuit potential difference (PD) method developed in our labwas used to evaluate compound activity at the ocular surface in vivo, asdepicted in FIG. 3A (21). Cl⁻ channel function was quantified bymeasuring PD during continuous perfusion of the ocular surface with aseries of solutions that imposed a transepithelial Cl⁻ gradient andcontained various channel agonists and/or inhibitors. The ocular surfacewas first perfused with isosmolar saline to record the baseline PD.Amiloride was then added to the perfusate, followed by exchange to a lowCl⁻ solution in which Cl⁻ with an impermeant anion, gluconate. Thesemaneuvers allow for direct visualization of CFTR activation in responseto addition of candidate CFTR activators.

FIG. 3B shows large hyperpolarizations following exposure toCFTR_(act)-B074, CFTR_(act)-J027 and CFTR_(act)-K089, which wereincreased relatively little by forskolin and were reversed byCFTR_(inh)-172. In comparison, VX-770 produced minimal changes in ocularsurface PD (FIG. 3C). FIG. 3D summarizes PD data for indicatedactivators, with data for additional compounds reported in FIG. 7.Control studies done in CF mice lacking functional CFTR showed nochanges in PD following addition of each of the compounds tested, with arepresentative curve shown for CFTR_(act)-K032 (FIG. 3E).

CFTR activators were next tested for their efficacy in augmenting tearproduction in mice. Preliminary experiments identified a standardophthalmic formulation (0.5% polysorbate) that increased compoundsolubility and duration-of-action. Following a single topical dose, theindirect CFTR activators cholera toxin, forskolin, and3-isobutyl-1-methylxanthine (IBMX) substantially increased basal tearsecretion at 30 min, but these effects were transient and undetectableafter 2 hours (FIG. 4A). However, the direct CFTR activators identifiedhere, CFTR_(act)-B074, CFTR_(act)-J027 and CFTR_(act)-K089, increasedtear fluid secretion by approximately two-fold for at least four hours.VX-770 produced little tear secretion (FIG. 4B). Repeated topicaladministrations (three times daily for up to 2 weeks) produced sustainedtear hypersecretion without tachyphylaxis (FIG. 4C). CFTR activators didnot increase tear fluid secretion in CF mice, demonstrating selectiveCFTR targeting (FIG. 4D).

Toxicity and Pharmacokinetics

Tear collection methods were validated by demonstrating reproduciblerecovery of tetramethylrhodamine dextran (3 kDa) from the ocular surfaceup to six hours after instillation. The pharmacokinetics ofCFTR_(act)-K089 at the ocular surface was determined by LC/MS ofrecovered tear washes. Following instillation of 0.1 nmol ofCFTR_(act)-K089 (2 μL, 50 μM) to the ocular surface, 7.9±2.4 pmol and0.011±0.004 pmol were recovered at five min and six hours, respectively(FIG. 5A). The amount of CFTR_(act)-K089 required for 50% CFTRactivation (EC₅₀˜250 nM) lies between the dashed lines, reflectingconcentrations calculated from the highest and lowest reported normaltear volumes in mice (33, 34). The quantity of CFTR_(act)-K089 recoveredfrom tear fluid predicts therapeutic levels for at least six hours. Tearfluid pharmacokinetics of CFTR_(act)-J027 could not be measured becausethe LC/MS sensitivity was low for this compound.

Following two weeks of three times per day dosing, the amounts ofCFTR_(act)-K089 and CFTR_(act)-J027 were below the limits of detection(˜10 and ˜700 fmol, respectively) in mouse blood, brain, liver andkidney, indicating minimal systemic accumulation. The chronicallytreated mice showed no signs of ocular toxicity, as assessed byslit-lamp evaluation for conjunctival hyperemia, anterior chamberinflammation, and lens clarity. LG staining showed no corneal orconjunctival epithelial disruption (FIG. 5B). The compounds alsoproduced no appreciable in vitro cytotoxicity in cell cultures atconcentrations up to 100 μM (FIG. 5C).

CFTR Activator Prevents Dry Eye in a Lacrimal Gland Excision Model inMice

On the basis of its favorable tear film pharmacokinetics,CFTR_(act)-K089 was selected for testing in a mouse model ofaqueous-deficient dry eye produced by LGE. Following extraorbital LGE inBALB/c mice, CFTR_(act)-K089-treated mice (0.1 nmol, administered threetimes daily) maintained basal tear volume, whereas tear volume fromvehicle-treated mice was significantly reduced at all subsequenttime-points (FIG. 6A), and for at least 30 days. Similar to what wasreported in C₅₇/b16 mice (30), decreased lacrimation in vehicle-treatedBALB/c mice was associated with progressive epithelial disruption fromDay 0 to Day 14, shown pictorially (FIG. 6B top) and quantitatively(FIG. 6C). CFTR_(act)-K089 not only restored tear secretion in LGE micebut remarkably prevented ocular surface epithelial disruption at alltime points (FIG. 6B bottom, C). Vehicle-treated eyes developed diffuse,progressive corneal epitheliopathy (LG score increase of 7.3±0.6 by Day14), whereas eyes treated with CFTR_(act)-K089 had minimal LG stainingat all time points (LG score change, −0.6±0.6).

Example 4—Constipation II

Abstract. Background & Aims: Constipation is a common clinical problemthat negatively impacts quality of life and is associated withsignificant health care costs. Activation of the cystic fibrosistransmembrane regulator (CFTR) chloride channel is the primary pathwaythat drives fluid secretion in the intestine, which maintainslubrication of luminal contents. We hypothesized that direct activationof CFTR would cause fluid secretion and reverse the excessivedehydration of stool found in constipation. Methods: A cell-basedhigh-throughput screen was done for 120,000 drug-like, synthetic smallmolecules. Active compounds were characterized for mechanism of actionand one lead compound was tested in a loperamide-induced constipationmodel in mice. Results: Several classes of novel CFTR activators wereidentified, one of which, the phenylquinoxalinone CFTR_(act)-J027, fullyactivated CFTR chloride conductance with EC₅₀˜200 nM, without causingelevation of cytoplasmic cAMP. Orally administered CFTR_(act)-J027normalized stool output and water content in a loperamide-induced mousemodel of constipation with ED₅₀˜0.5 mg/kg; CFTR_(act)-J027 was withouteffect in cystic fibrosis mice lacking functional CFTR. Short-circuitcurrent, fluid secretion and motility measurements in mouse intestineindicated a pro-secretory action of CFTR_(act)-J027 without directstimulation of intestinal motility. Oral administration of 10 mg/kgCFTR_(act)-J027 showed minimal bioavailability, rapid hepatic metabolismand blood levels <200 nM, and without apparent toxicity after chronicadministration. Conclusions: CFTR_(act)-J027 or alternativesmall-molecule CFTR-targeted activators may be efficacious for thetreatment of constipation.

Introduction

Constipation is a common clinical complaint in adults and children thatnegatively impacts quality of life. The prevalence of chronicconstipation has been estimated to be 15% in the US population, withannual health-care costs estimated at ˜7 billion dollars with >800million dollars spent on laxatives [1, 2]. The mainstay of constipationtherapy includes laxatives that increase stool bulk, such as solublefiber; create an osmotic load, such as polyethylene glycol; or stimulateintestinal contraction, such as the diphenylmethanes. There are alsosurface laxatives that soften stool such as docusate sodium andprobiotics such as Lactobacillus paracasei [3]. The FDA-approved druglinaclotide, a peptide agonist of the guanylate cyclase C receptor, actsby inhibiting visceral pain, stimulating intestinal motility, andincreasing intestinal secretion [4, 5]. A second approved drug,lubiprostone, a prostaglandin E analog, is thought to activate aputative enterocyte CIC-2 channel [6], though the mechanistic data areless clear. Despite the wide range of therapeutic options, there is acontinued need for safe and effective drugs to treat constipation.

Intestinal fluid secretion involves active Cl⁻ secretion across theenterocyte epithelium through the basolateral membrane Na⁺/K⁺/2Cl⁻cotransporter (NKCC1) and the luminal membrane cystic fibrosistransmembrane regulator (CFTR) Cl⁻ channel and Ca²⁺-activated Cl⁻channel (CaCC). The electrochemical and osmotic forces created by Cl⁻secretion drive Na⁺ and water secretion [7]. In cholera and Traveler'sdiarrhea CFTR is strongly activated by bacterial enterotoxins throughelevation of intracellular cyclic nucleotides [8, 9]. CFTR is anattractive target to increase intestinal fluid secretion in constipationas it is robustly expressed throughout the intestine and its activationstrongly increases intestinal fluid secretion. An activator targetingCFTR directly is unlikely to produce the massive, uncontrolledintestinal fluid secretion seen in cholera because the enterotoxins incholera act irreversibly to produce sustained elevation of cytoplasmiccAMP, which not only activates CFTR but also basolateral K⁺ channels,which increase the electrochemical driving force for Cl⁻ secretion;cholera enterotoxins also inhibit the luminal NHE3 Na⁺/H⁺ exchangerinvolved in intestinal fluid absorption [10, 11].

Motivated by these considerations and the continuing need for safe andeffective drug therapy of constipation, here we report theidentification and characterization of a nanomolar-potency,CFTR-targeted small-molecule activator, and provide proof of concept forits pro-secretory action in intestine and efficacy in constipation.

Methods.

Materials. High-throughput screening was done using a diverse collectionof 120,000 drug-like synthetic compounds obtained from ChemDiv Inc. (SanDiego, Calif., USA) and Asinex (Winston-Salem, N.C., USA). Forstructure-activity analysis, 600 commercially available analogs (ChemDivInc.) of active compounds identified in the primary screen were tested.Other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA)unless indicated otherwise.

CFTR_(act)-J027 synthesis. To a solution of o-phenylenediamine (1 g,9.24 mmol) in DMF (30 mL) was added potassium carbonate (2.5 g, 18.4mmol) and benzyl bromide (0.73 mL, 6.2 mmol) then stirred overnight atambient temperature. The reaction mixture was diluted with CH₂Cl₂,washed with water, dried over MgSO₄ and concentrated under reducedpressure. The residue was purified by flash chromatography to give theintermediate N¹-benzylbenzene-1,2-diamine as a brown liquid. ¹H NMR (300MHz, CDCl₃): δ 7.45-7.31 (m, 5H), 6.86-6.69 (m, 4H), 4.35 (s, 2H), 3.50(br, 3H); MS: m/z 199 (M+H). Then, a solution of the intermediate (400mg, 2 mmol) and 5-nitroisatin (380 mg, 2 mmol) in acetic acid (5 mL) wasrefluxed for 2 h. The reaction mixture was cooled to room temperatureand solvent removed under reduced pressure. The residue was dissolvedwith methanol and acetic acid was added to crystallize3-(2-amino-5-nitrophenyl)-1-benzylquinoxalin-2(1H)-one (CFTR_(act)-J027)as a yellow powder with >99% purity. ¹H NMR (300 MHz, DMSO-d6): δ 9.15(d, 1H, J=2.8 Hz), 8.07 (dd, 1H, J=2.7, 9.2 Hz), 7.97 (dd, 1H, J=1.2,7.9 Hz), 7.82 (brs, 2H), 7.60-7.27 (m, 7H), 6.92 (d, 1H, J=9.2 Hz), 5.59(brs, 2H); ¹³C NMR (75 MHz, DMSO-d6): δ 155.0, 154.6, 153.3, 136.3,135.3, 132.8, 132.2, 131.0, 130.0, 129.5, 129.1, 127.7, 127.3, 126.8,124.1, 116.1, 115.9, 115.4, 45.9; MS: m/z 373 (M+H).

Cell culture. Fischer Rat Thyroid (FRT) cells stably co-expressing humanwild-type CFTR and the halide-sensitive yellow fluorescent protein(YFP)-H148Q were generated as previously described [12]. Cells werecultured on plastic in Coon's-modified Ham's F12 medium supplementedwith 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin,and 100 □g/ml streptomycin. For high-throughput screening, cells wereplated in black 96-well microplates (Corning-Costar Corp., Corning,N.Y., USA) at a density of 20,000 cells per well. Screening was done24-48 hours after plating.

High-throughput screening. Screening was carried out using a BeckmanCoulter integrated system equipped with a liquid handling system and twoFLUOstar fluorescence plate readers (BMG Labtechnologies, Durham, N.C.,USA), each equipped with dual syringe pumps and 500±10 nm excitation and535±15 nm emission filters (details in ref. 12). CFTR- andYFP-expressing FRT cells were grown at 37° C./5% CO₂ for 24-48 hoursafter plating. At the time of assay, cells were washed three times withphosphate-buffered saline (PBS) and then incubated for 10 min with 60 μlof PBS containing test compounds (at 10 μM) and a low concentration offorskolin (125 nM). Each well was assayed individually for I⁻ influx ina plate reader by recording fluorescence continuously (200 ms per point)for 2 s (baseline) and then for 12 s after rapid (<1 s) addition of 165μL of PBS in which 137 mM Cl⁻ was replaced by I⁻. The initiate rate ofI⁻ influx was computed by determined using exponential regression. Allcompound plates contained negative controls (DMSO vehicle) and positivecontrols (20 □M forskolin).

Short-circuit current measurement. Short-circuit current was measured inFRT cells stably expressing wild-type human CFTR cultured on porousfilters as described [12]. The basolateral solution contained 130 mMNaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1 mM CaCl₂, 0.5 mM MgCl₂, 10 mMglucose, and 10 mM Na-HEPES (pH 7.3, 37° C.). In the apical solution 65mM NaCl was replaced by Na gluconate, and CaCl₂ was increased to 2 mM,and the basolateral membrane was permeabilized with 250 μg/mlamphotericin B. Short-circuit current was measured in freshly harvestedadult mouse colon at 37° C. using symmetrical Krebs-bicarbonate buffer.

cAMP assay. Intracellular cAMP activity was measured using a GloSensorluminescence assay (Promega Corp., Madison, Wis., USA). FRT null cellswere stably transfected with the pGloSensor cAMP plasmid and plated ontowhite 96-well microplates and grown to confluence. Cells were washedthree times with PBS and incubated with 5 μM CFTR_(act)-J027 for 10 minin the absence and presence of 100 nM forskolin. cAMP was assayedaccording to the manufacturer's instructions.

Pharmacokinetics. All animal experiments were approved by UCSFInstitutional Animal Care and Use Committee. Female CD1 mice weretreated with 10 mg/kg CFTR_(act)-J027 (saline containing 5% DMSO and 10%Kolliphor HS 15) either intraperitoneally (ip) or orally. Blood wascollected at 15, 30, 60, 150, 240 and 360 min after treatment by orbitalpuncture and centrifuged at 5000 rpm for 15 min to separate plasma.Plasma samples (60 μL) were mixed with 300 μL acetonitrile andcentrifuged at 13000 rpm for 20 min, and 90 μL of the supernatant wasused for LC/MS. The solvent system consisted of a linear gradient from 5to 95% acetonitrile over 16 min (0.2 ml/min flow). Mass spectra wasacquired on a mass spectrometer (Waters 2695 and Micromass ZQ) usingelectrospray (+) ionization, mass ranging from 100 to 1500 Da, conevoltage 40 V. Calibration standards were prepared in plasma fromuntreated mice to which known amounts of CFTR_(act)-J027 were added.

In vitro metabolic stability. CFTR_(act)-J027 (5 μM) was incubated forspecified times at 37° C. with mouse liver microsomes (1 mg protein/ml;Sigma-Aldrich) in potassium phosphate buffer (100 mM) containing 1 mMNADPH, as described [13]. The mixture was then chilled on ice, and 0.5ml of ice-cold ethyl acetate was added. Samples were centrifuged for 15min at 3000 rpm, the supernatant evaporated to dryness, and the residuewas dissolved in 100 μL mobile phase (acetonitrile:water, 3:1) for LC/MSand assayed as described above.

Murine model of constipation. Female CD1 mice (age 8-10 weeks) wereadministered loperamide (0.3 mg/kg, ip, Sigma-Aldrich) to produceconstipation. Various amounts of CFTR_(act)-J027 (0.1, 0.3, 1, 3 and 10mg/kg) were given at the same time (for ip administration) or 1 h before(for oral administration) loperamide. Control mice were treated withvehicle only. Some mice were treated orally with lubiprostone (0.5mg/kg, Sigma-Aldrich) or linaclotide (0.5 mg/kg, Toronto ResearchChemicals Inc., Toronto, Ontario, Canada). After loperamide injection,mice were placed individually in metabolic cages with food and waterprovided ad libitum. Stool samples were collected for 3 h, and totalstool weight and number of fecal pellets were quantified. To measurestool water content stool samples were dried at 80° C. for 24 h andwater content was calculated as [wet weight-dry weight]/wet weight.Similar studies were done in cystic fibrosis (CF) mice (ΔF508homozygous) lacking functional CFTR. Some studies were done using thechemically similar but inactive analog of CFTR_(act)-J027,3-(2-amino-5-nitrophenyl)-1-(methyl)-2(1H)-quinoxalinone.

In vivo intestinal transit and ex vivo intestinal contractility.Whole-gut transit time was determined using an orally administeredmarker (200 μL, 5% Evans Blue, 5% gum Arabic) and measuring the time ofits appearance in stool. Mice were administered loperamide andCFTR_(act)-J027 (10 mg/kg) or vehicle intraperitoneally at zero time.For ex vivo contractility measurements, mice were euthanized by avertinoverdose (200 mg/kg, 2,2,2-tribromethanol, Sigma-Aldrich) and ileum andcolon segments of ˜2 cm length were isolated and washed withKrebs-Henseleit buffer. The ends of the intestinal segments were tied,connected to a force transducer (Biopac Systems, Goleta, Calif., USA)and tissues were transferred to an organ chamber (Biopac Systems)containing Krebs-Henseleit buffer at 37° C. aerated with 95% O₂, 5% CO₂.Ileum and colon were stabilized for 60 min with resting tensions of 0.5and 0.2 g respectively, and solutions were changed every 15 min. Effectsof CFTR_(act)-J027 on baseline and loperamide-suppressed isometricintestinal contractions were recorded.

In vivo intestinal secretion and absorption. Mice (wildtype or CF) weregiven access to 5% dextrose water but not solid food for 24 h beforeexperiments. Mice were anesthetized with isoflurane and body temperaturewas maintained during surgery at 36-38° C. using a heating pad. A smallabdominal incision was made to expose the small intestine, and closedmid-jejunal loops (length 2-3 cm) were isolated by sutures. Loops wereinjected with 100 μL vehicle alone or 100 μg CFTR_(act)-J027 in vehicle.The abdominal incision was closed with sutures, and mice were allowed torecover from anesthesia. Intestinal loops were removed at 90 min andloop length and weight were measured to quantify fluid secretion.Intestinal absorption was measured in CF mice (to prevent secretion) asdescribed above, except that the loops were removed at 0 or 30 min.Absorption was calculated as 1-(loop weight at 0 min−loop weight at 30min)/loop weight at 0 min.

Chronic administration and toxicity studies. Mice were administered 10mg/kg CFTR_(act)-J027 or vehicle orally once a day for 7 d. One hourafter the final dose mice were treated with loperamide (0.3 mg/kg, ip)and stool was collected for 3 h. In vivo toxicity was assessed in thesemice by measuring lung wet/dry weight ratio, complete blood count(HEMAVET 950FS, Drew Scientific Inc., Florida, USA) and serum chemistry(Idexx Laboratories Inc., Sacramento, Calif., USA) 4 h after the lastCFTR_(act)-J027 dose. In vitro cytotoxicity was measured in FRT cellsincubated with 25 □M CFTR_(act)-J027 for 8 and 24 h. Cytotoxicity wasmeasured by Alamar Blue assay according to the manufacturer'sinstructions (Invitrogen, Carlsbad, Calif., USA).

Statistical analysis. Experiments with two groups were analyzed withStudent's t-test, when there are 3 groups or more analysis was made withone-way analysis of variance and post-hoc Newman-Keuls multiplecomparisons test. P<0.05 was taken as statistically significant.

Results.

Identification and in vitro characterization of small-molecule CFTRactivators. The goal was to identify a potent, CFTR-targeted activatorwith pro-secretory activity in intestine in order test its efficacy in amouse model of constipation. FIG. 8A summarizes the project strategy.The compounds evaluated here included small molecules identified inprior CFTR activator/potentiator screens [14] and from a new screen ofsynthetic small molecules not tested previously. The most activecompounds emerging from the screen, along with commercially availablechemical analogs, were prioritized based on an initial mechanism ofaction study (assay of cAMP elevation), in vitro toxicity, pro-secretoryaction in mouse intestine, and efficacy in a mouse model ofconstipation. FIG. 8B shows the cell-based plate reader screening methodin which the initial rate of iodide influx was measured in FRT cellsstably expressing human wildtype CFTR and a YFP fluorescent halidesensor following extracellular iodide addition. A CFTR activatorincreases the initial slope of the fluorescence quenching curve.

FIG. 8C shows chemical structures of six classes of CFTR candidateactivators identified from the screens. Based on the criteria listedabove, we focused further studies on CFTR_(act)-J027, a3-phenyl-quinoxalinone with drug-like properties. CFTR_(act)-J027 wassynthesized in pure crystalline form in two steps (FIG. 8D).

Short-circuit current measurements in CFTR-expressing FRT cells showedthat CFTR_(act)-J027 fully activated CFTR (FIG. 9A), as the cAMP agonistforskolin produced no further increase in current, with an EC₅₀˜200 nM(FIG. 9B). Interestingly, CFTR_(act)-J027 was only a weak potentiator ofΔF508-CFTR, as studied in FRT cells expressing ΔF508-CFTR afterovernight incubation with a corrector (FIG. 9C). Cl⁻ secretion infreshly isolated mouse colon showed a concentration-dependent increasein short-circuit current with EC₅₀˜300 nM (FIG. 9D). The increase incurrent at high CFTR_(act)-J027 was further increased by forskolin,which may be a consequence of activation of a basolateral membranecAMP-sensitive K⁺ channel that increases the driving force for apicalmembrane Cl⁻ secretion. The increase in current was fully inhibited by aCFTR-selective inhibitor. FIG. 9E shows that CFTR_(act)-J027 does notelevate cellular cAMP when added alone, and does not further increasecAMP when added together with forskolin, suggesting that CFTR activationinvolves a direct interaction mechanism rather than indirect actionthrough cAMP elevation.

CFTR_(act)-J027 normalizes stool output in a mouse model ofconstipation. CFTR_(act)-J027 was studied in the well-establishedloperamide-induced mouse model of constipation in which stool weight,pellet number and water content were measured over 3 h followingintraperitoneal loperamide administration (FIG. 10A). Intraperitonealadministration of CFTR_(act)-J027 at 10 mg/kg normalized each of thestool parameters. CFTR_(act)-J027 did not affect stool output or watercontent in control (non-loperamide-treated) mice. Importantly,CFTR_(act)-J027 was without effect in cystic fibrosis mice lackingfunctional CFTR (FIG. 10B), nor was an inactive chemical analog ofCFTR_(act)-J027 effective in wildtype mice (FIG. 10C). These resultssupport a CFTR-selective action of CFTR_(act)-J027. Dose-responsestudies in mice showed an ED₅₀ of 2 mg/kg in the loperamide model by ipadministration of CFTR_(act)-J027 (FIG. 10D).

Oral administration of 10 mg/kg CFTR_(act)-J027 1 h prior to loperamideadministration was also effective in normalizing stool output and watercontent in loperamide-treated mice, with no effect in control mice (FIG.11A). The ED₅₀ for oral administration was 0.5 mg/kg, substantiallylower than that for ip administration (FIG. 11B). In parallel studies,oral administration of the approved drugs lubiprostone or linaclotide at250-500 fold greater mg/kg doses than given to humans for treatment ofconstipation, were less effective in normalizing stool output, producing50% and 35% of the maximal CFTR_(act)-J027 response, respectively (FIG.11C).

CFTR_(act)-J027 actions on intestinal transit, motility and fluidtransport. CFTR_(act)-J027 action on intestinal transit and motility wasmeasured in vivo and in isolated intestinal strips, respectively.Whole-gut transit time, as measured by appearance of a marker in thestool after bolus oral gavage at the time of ip loperamide andCFTR_(act)-J027 administration, was normalized by CFTR_(act)-J027 (FIG.12A, left panel). CFTR_(act)-J027 had no effect on whole-gut transittime in cystic fibrosis mice (right panel). In vitro measurements ofintestinal contraction showed no effect of CFTR_(act)-J027 added aloneor in the presence of 10 μM loperamide in isolated mouse ileum and colonstrips (FIG. 12B). CFTR_(act)-J027 may thus increase intestinal transitin vivo by stimulating motility by secretion-induced stretch of the gutwall, without direct effect on intestinal smooth muscle.

To directly investigate the effects of CFTR_(act)-J027 on intestinalfluid secretion and absorption, an in vivo closed-intestinal loop modelwas used. CFTR_(act)-J027 was injected into closed, mid-jejunal loopsand fluid accumulation was measured at 90 min. CFTR_(act)-J027 produceda 140% increase in loop weight/length ratio, indicating fluid secretioninto the intestinal lumen in wild-type mice (FIG. 12C, upper panel), butwas without effect in cystic fibrosis mice (lower panel), supporting aCFTR-selective mechanism of action. A closed-loop model was also used tostudy CFTR_(act)-J027 action on intestinal fluid absorption. Fluidwithout or with CFTR_(act)-J027 was injected into closed, mid-jejunalloops of cystic fibrosis mice (to avoid confounding fluid secretion) andfluid absorption was measured at 30 min. CFTR_(act)-J027 did not affectintestinal fluid absorption (FIG. 12D).

CFTR_(act)-J027 pharmacology and toxicity in mice. The in vitrometabolic stability of CFTR_(act)-J027 was measured by incubation withmouse liver microsomes in the presence of NADPH. CFTR_(act)-J027 wasrapidly metabolized with ˜21 min elimination half-life, with only 7% ofthe original compound remaining at 60 min (FIG. 13A).

Pharmacokinetics was measured in mice following bolus intraperitoneal ororal administration of 10 mg/kg CFTR_(act)-J027. Following ipadministration serum CFTR_(act)-J027 concentration decreased with anelimination half-life of ˜16 min, and was undetectable at 150 min (FIG.13B). Following oral administration serum CFTR_(act)-J027 concentrationreached 180 nM at 30 min and was undetectable at other time points (FIG.13B).

Preliminary toxicological studies of CFTR_(act)-J027 were done in cellcultures and mice. CFTR_(act)-J027, at a concentration of 20 μM near itssolubility limit, did not show cytotoxicity as measured by the AlamarBlue assay (FIG. 13C). In the 7-day treated mice, CFTR_(act)-J027 didnot affect the major serum chemistry and blood parameters (Table 1,Example 1), nor did it change body weight or produce airway/lung fluidaccumulation (FIG. 13D).

Last, to determine whether chronically administered CFTR_(act)-J027retained efficacy, mice were treated orally for 7 days with 10 mg/kgCFTR_(act)-J027 or vehicle, and loperamide was given 1 h after the finaldose. FIG. 13E shows that chronically administered CFTR_(act)-J027remained effective in normalizing stool output and water contentfollowing loperamide.

Discussion

We identified by high-throughput screening a nanomolar-affinity,small-molecule CFTR activator, CFTR_(act)-J027, and demonstrated itspro-secretory action in mouse intestine and its efficacy in normalizingstool output in a loperamide-induced mouse model of constipation.Constipation remains a significant clinical problem in outpatient andhospitalized settings. Opioid-induced constipation is a common adverseeffect in patients after surgery, undergoing chemotherapy and withchronic pain.

CFTR-targeted activation adds to the various mechanisms of action ofanti-constipation therapeutics. It is notable that pure CFTR activationis able to produce a robust Cl⁻ current and fluid secretion response inthe intestine, without causing global elevation of cyclic nucleotideconcentration, direct stimulation of intestinal contractility, oralteration of intestinal fluid absorption. Linaclotide, a peptideagonist of the guanylate cyclase C receptor that increases intestinalcell cGMP concentration. Linaclotide inhibits activation of colonicsensory neurons and activates motor neurons, which reduces pain andincreases intestinal smooth muscle contraction; in addition, elevationin cGMP concentration in enterocytes may activate CFTR and have apro-secretory action [4, 5]. A second approved drug, the prostaglandin Eanalog lubiprostone, is thought to activate a putative enterocyte CIC-2channel [6], though the mechanistic data are less clear. Compared withthese drugs, a pure CFTR activator has a single, well-validatedmechanism of action and does not produce a global cyclic nucleotideresponse in multiple cell types. Of note, linaclotide and lubiprostoneshowed limited efficacy in clinical trials. Linaclotide was effective in˜20% of chronic constipation patients of whom ˜5% also responded toplacebo [15], and lubiprostone was effective in ˜13% of IBS-C patientsof whom ˜7% responded to placebo [16]. Based on our mouse data showingsubstantially greater efficacy of CFTR_(act)-J027 compared tosupramaximal doses of linaclotide or lubiprostone, we speculate thatCFTR activators may have greater efficacy in clinical trials.

CFTR_(act)-J027 is substantially more potent for activation of wildtypeCFTR than VX-770 (ivacaftor), the FDA-approved drug for treatment ofcystic fibrosis (CF) caused by certain CFTR gating mutations. In FRTcells expressing wild-type CFTR, short-circuit current measurementshowed nearly full activation of CFTR by CFTR_(act)-J027 at 3 □M whereasVX-770 maximally activated CFTR by only 15%. However, CFTR_(act)-J027was substantially less potent than ivacaftor as a ‘potentiator’ ofdefective chloride channel gating of the most common CF-causingmutation, ΔF508, which is not unexpected, as potentiator efficacy in CFis mutation-specific. In addition to its potential therapeutic utilityfor constipation, a small-molecule activator of wildtype CFTR may beuseful for treatment of chronic obstructive pulmonary disease andbronchitis, asthma, cigarette smoke-induced lung dysfunction, dry eyeand cholestatic liver disease [17-19].

Substituted quinoxalinones were reported as selective antagonists of themembrane efflux transporter multiple-drug-resistance protein 1 [20].Quinoxalinones have also been reported to show anti-diabetic activity bystimulating insulin secretion in pancreatic INS-1 cells [21], andinhibitory activity against serine proteases for potential therapy ofthrombotic disorders [22]. Recently, quinoxalinones have been reportedto inhibit aldose reductase [23]. These reports suggest that thequinoxalinone scaffold has drug-like properties. Synthetically,quinoxalinone can be prepared in one to four steps from commerciallyavailable starting materials [24], which allows facile synthesis oftargeted analogs.

In addition to compound-specific off-target actions, the potentialside-effects profile of a CFTR activator could include pro-secretoryactivity in the airway/lungs and various glandular and other epithelia.Off-target effects for constipation therapy could be limited by oraladministration of a CFTR activator with limited intestinal absorptionand/or rapid systemic clearance to minimize systemic exposure.CFTR_(act)-J027 when administered orally at a high dose (10 mg/kg)showed very low bioavailability with blood levels well below the EC₅₀for CFTR activation, which may be due to first-pass effect as evidencedits rapid in vitro metabolism in liver microsomes. CFTR_(act)-J027 didnot show significant in vitro cytotoxicity at a concentration of 25μM, >100-fold greater than its EC₅₀ for CFTR activation, or in vivotoxicity in mice in a 7-day study at a maximal efficacious dose thatnormalized stool output in the loperamide model of constipation. Thepotentially most significant off-target action, stimulation oflung/airway fluid secretion, was not seen as evidenced by normal lungwater content in the 7-day treated mice. These limited toxicity studiesoffer proof of concept for application of a CFTR activator inconstipation.

In summary, without wishing to be bound by theory, it is believed thatthe data herein provide evidence for the pro-secretory action of a CFTRactivator in mouse intestine and proof of concept for its use intreatment of various types of constipation, which could includeopioid-induced constipation, chronic idiopathic constipation, andirritable bowel syndrome with constipation predominance.

References (Example 4)

-   [1]. Pinto Sanchez M I, Bercik P. Epidemiology and burden of chronic    constipation. Canadian Journal of Gastroenterology 2011, 25(Suppl    B): 11B-15B; [2]. Mugie S M, Di Lorenzo C, Benninga M A.    Constipation in childhood. Nature Reviews Gastroenterology and    Hepatology 2011, 8(9):502-511; [3]. Menees S, Saad R, Chey W D.    Agents that act luminally to treat diarrhoea and constipation.    Nature Reviews Gastroenterology and Hepatology 2012, 9(11):661-674;    [4]. Castro J, Harrington A M, Hughes P A et al. Linaclotide    inhibits colonic nociceptors and relieves abdominal pain via    guanylate cyclase-C and extracellular cyclic guanosine    3′,5′-monophosphate. Gastroenterology 2013, 145(6): 1334-1346; [5].    Busby R W, Bryant A P, Bartolini W P et al. Linaclotide, through    activation of guanylate cyclase C, acts locally in the    gastrointestinal tract to elicit enhanced intestinal secretion and    transit. European Journal of Pharmacology 2010, 649(1-3):328-335;    [6]. Fei G, Raehal K, Liu S et al. Lubiprostone reverses the    inhibitory action of morphine on intestinal secretion in Guinea pig    and mouse. Journal of Pharmacology and Experimental Therapeutics    2010, 334(1):333-340; [7]. Thiagarajah J R, Donowitz M, Verkman A S.    Secretory diarrhoea: mechanisms and emerging therapies. Nature    Reviews Gastroenterology and Hepatology 2015, 12(8):446-457; [8].    Field M, Fromm D, Al-Awqati Q et al. Effect of cholera enterotoxin    on ion transport across isolated ileal mucosa. The Journal of    Clinical Investigation 1972, 51(4):796-804; [9]. Rao M C, Guandalini    S, Smith P L et al. Mode of action of heat-stable Escherichia coli    enterotoxin Tissue and subcellular specificities and role of cyclic    GMP. Biochimica et Biophysica Acta (BBA)—General Subjects 1980,    632(1):35-46; [10]. Subramanya S B, Rajendran V M, Srinivasan P et    al. Differential regulation of cholera toxin-inhibited Na—H exchange    isoforms by butyrate in rat ileum. American Journal of    Physiology—Gastrointestinal and Liver Physiology 2007,    293(4):G857-G863; [11]. Hecht G, Hodges K, Gill R K et al.    Differential regulation of Na⁺/H⁺ exchange isoform activities by    enteropathogenic E. coli in human intestinal epithelial cells.    American Journal of Physiology—Gastrointestinal and Liver Physiology    2004, 287(2):G370-G378; [12]. Galietta L J V, Springsteel M F, Eda M    et al. Novel CFTR chloride channel activators identified by    screening of combinatorial libraries based on flavone and    benzoquinolizinium lead compounds. Journal of Biological Chemistry    2001, 276(23): 19723-19728; [13]. Esteva-Font C, Cil O, Phuan P W et    al. Diuresis and reduced urinary osmolality in rats produced by    small-molecule UT-A-selective urea transport inhibitors. The FASEB    Journal 2014, 28(9):3878-3890; [14]. Ma T, Vetrivel L, Yang H et al.    High-affinity activators of cystic fibrosis transmembrane    conductance regulator (CFTR) chloride conductance identified by    high-throughput screening. Journal of Biological Chemistry 2002,    277(40):37235-37241; [15]. Lembo A J, Schneier H A, Shiff S J et al.    Two randomized trials of linaclotide for chronic constipation. New    England Journal of Medicine 2011, 365(6):527-536; [16]. Website:    www.amitizahcp.com; [17]. Gras D, Chanez P, Vachier I et al.    Bronchial epithelium as a target for innovative treatments in    asthma. Pharmacology & Therapeutics 2013, 140(3):290-305; [18].    Srivastava A. Progressive familial intrahepatic cholestasis. Journal    of Clinical and Experimental Hepatology 2014, 4(1):25-36; [19].    Levin M H, Verkman A S. CFTR-regulated chloride transport at the    ocular surface in living mice measured by potential differences.    Investigative Ophthalmology & Visual Science 2005, 46(4): 1428-1434;    [20]. Lawrence D S, Copper J E, Smith C D. Structure-activity    studies of substituted quinoxalinones as multiple-drug-resistance    antagonists. Journal of Medicinal Chemistry 2001, 44(4):594-601;    [21]. Botton G, Valeur E, Kergoat M et al. Preparation of    quinoxalinone derivatives as insulin secretion stimulators useful    for the treatment of diabetes. PCT Int Appl 2009, WO 2009109258 A1    20090911 (patent); [22]. Dudley D A, Edmunds J J. Preparation of    quinoxalinones as serine protease inhibitors for treatment of    thrombotic disorders. PCT Int Appl 1999:WO 9950254 A9950251 19991007    (patent); [23]. Qin X, Hao X, Han H et al. Design and Synthesis of    potent and multifunctional aldose reductase inhibitors based on    auinoxalinones. Journal of Medicinal Chemistry 2015, 58(3):    1254-1267; [24]. Shaw A D, Denning C R, Hulme C. One-pot two-step    synthesis of quinoxalinones and diazepinones via a tandem oxidative    amidation-deprotection-cyclization sequence. Synthesis 2013,    45(4):459-462.

Example 5—Dry Eye—II

Abbreviations: CFTR, cystic fibrosis transmembrane conductanceregulator; cAMP, cyclic adenosine monophosphate; ENaC, epithelial sodiumchannel; YFP, yellow fluorescent protein; CF, cystic fibrosis; FRTcells, Fischer rat thyroid cells; I_(SC), short-circuit current; PD,potential difference; IBMX, 3-isobutyl-1-methylxanthine; fsk, forskolin;LC/MS, liquid chromatography/mass spectroscopy; LG, lissamine green;LGE, lacrimal gland excision.

Abstract. Dry eye disorders, including Sjögren's syndrome, constitute acommon problem in the aging population with limited effectivetherapeutic options available. The cAMP-activated Cl— channel CFTR(cystic fibrosis transmembrane conductance regulator) is a majorpro-secretory chloride channel at the ocular surface. Here, weinvestigated whether compounds that target CFTR can correct the abnormaltear film in dry eye. Small-molecule activators of human wild-type CFTRidentified by high-throughput screening were evaluated in cell cultureand in vivo assays to select compounds that stimulate Cl—-driven fluidsecretion across the ocular surface in mice. Anaminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cellcultures with EC50˜250 nM and produced a ˜8.5 mV hyperpolarization inocular surface potential difference. When delivered topically,CFTRact-K089 doubled basal tear secretion for four hours and had noeffect in CF mice. CFTRact-K089 showed sustained tear filmbioavailability without detectable systemic absorption. In a mouse modelof aqueous-deficient dry eye produced by lacrimal gland excision,topical administration of 0.1 nmol CFTRact-K089 three times dailyrestored tear secretion to basal levels and fully prevented the cornealepithelial disruption seen in vehicle-treated controls. Our resultssupport potential utility of CFTR-targeted activators as a novelpro-secretory treatment for dry eye.

Introduction

Dry eye is a heterogeneous group of disorders with common features ofreduced tear volume and tear fluid hyperosmolarity, which lead toinflammation at the ocular surface. The clinical consequences, whichinclude eye discomfort and visual disturbance, represent a major publichealth concern in an aging population. Dry eye affects up to one-thirdof the global population (1), including five million Americans age 50and over (2, 3). The economic burden of dry eye is substantial, withdirect annual health care costs estimated at $3.84 billion dollars inthe United States (4).

Ninety-four percent of surveyed ophthalmologists believe that additionaltreatments are needed for moderate-to-severe dry eye (7).

The ocular surface is a collection of anatomically continuous epithelialand glandular tissues that are functionally linked to maintain the tearfilm (8). While lacrimation contributes the bulk of reflex tearing, thecomea and conjunctiva regulate basal tear volume and composition. Theprincipal determinants of water movement across the ocular surface intothe tear film include apical chloride (Cl⁻) secretion through cAMP- andcalcium (Ca²⁺)-dependent Cl⁻ transporters, and sodium (Nat) absorptionlargely though the epithelial Na⁺ channel (ENaC).

The cystic fibrosis transmembrane conductance regulator (CFTR) is acAMP-activated Cl⁻ channel expressed in some secretory epithelial cells,including those in cornea and conjunctiva (14-16). We found substantialcapacity for active CFTR-facilitated Cl⁻ at the ocular surface in mice(21, 22), as subsequently shown in rat conjunctiva (23), providing arational basis for investigation of CFTR activators as a pro-secretorystrategy for dry eye. The only clinically approved CFTR activator,VX-770 (ivacaftor), is indicated for potentiating the channel gating ofcertain CFTR mutants causing CF, but only weakly activates wild-typeCFTR (24, 25).

Here, we evaluated and prioritized novel small-molecule activators ofwild-type CFTR identified by high-throughput screening as potentialtopical therapy for dry eye, with the research strategy summarized inFIG. 1. The goal was to improve upon our previously identified CFTRactivators (26), which lack suitable potency and chemical properties tobe advanced to clinical development, and to demonstrate efficacy ofnewly identified CFTR activator(s) in a mouse model of dry eye.

Materials and Methods.

Mice. Wild-type (WT) and CF (homozygous ΔF508-CFTR mutant) mice in a CD1genetic background were bred at the University of California SanFrancisco (UCSF) Animal Facility. Mice aged 8 to 12 weeks (25 to 35 g)were used. Female BALB/c mice (7-8 weeks old) were purchased from theHarlan Laboratory (Livermore, Calif., USA). Animal protocols wereapproved by the UCSF Institutional Animal Care and Use Committee andwere in compliance with the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Short-circuit current. Fischer rat thyroid (FRT) cells stably expressingwild-type human CFTR were cultured on Snapwell inserts (Corning Costar,Corning N.Y., USA) for short-circuit current (I_(sc)) measurements.After 6-9 days in culture, when the transepithelial resistance was >1000Ω/cm², the inserts were mounted in an Ussing chamber system (WorldPrecision Instruments, Sarasota, Fla., USA). The basolateral solutioncontained 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 1 mM CaCl₂, 0.5 mMMgCl₂, 10 mM glucose, and 10 mM Na-HEPES (pH 7.3). In the apical bathingsolution, 65 mM NaCl was replaced by Na gluconate, and CaCl₂ wasincreased to 2 mM. Both solutions were bubbled with air and maintainedat 37° C. The basolateral membrane was permeabilized with 250 μg/mlamphotericin B (26, 27). Hemichambers were connected to a DVC-1000voltage clamp via Ag/AgCl electrodes and 3 M KCl agar bridges for I_(sc)recording.

cAMP and cytotoxicity assays. Intracellular cAMP activity was measuredusing a GloSensor luminescence assay (Promega Corp., Madison, Wis.,USA). FRT cells stably transfected with the pGloSensor cAMP plasmid(Promega Corp.) were cultured in white 96-well microplates (CorningCostar) overnight. Cells were then washed three times with PBS andincubated with 5 μM test compound for 10 min in the absence and presenceof 100 nM forskolin. To assay cytotoxicity, FRT cells were culturedovernight in black 96-well Costar microplate wells and incubated withtest compounds at up to 100 μM (the maximum solubility in PBS) for 1 or24 h. Cytotoxicity was measured by Alamar Blue assay according to themanufacturer's instructions (Invitrogen, Carlsbad, Calif., USA).

Ocular surface potential difference measurements. Open-circuittransepithelial PD were measured continuously in anesthetized mice inresponse to serial perfusions of different solutions over the ocularsurface, as described (21). Mice were anesthetized with Avertin(2,2,2-tribromoethanol, 125 mg/kg intraperitoneal, Sigma-Aldrich, St.Louis, Mo., USA), and core temperature was maintained at 37° C. using aheating pad. Eyes were oriented with the comea and conjunctiva facingupward and exposed by retracting the eyelid with cross-action forceps.Solutions were isosmolar (320±10 mOsM; compositions provided in ref. 21)and contained 10μ□□ indomethacin to prevent CFTR activation byprostaglandins. The ocular surface was perfused at 6 mL/min throughplastic tubing using a multireservoir gravity pinch-valve system (ALAScientific, Westbury, N.Y., USA) and variable-flow peristaltic pump(medium flow model; Fisher Scientific, Fair Lawn, N.J., USA). A probecatheter was fixed 1 mm above the comea using a micropositioner and asuction cannula was positioned 3 mm from the orbit. The measuringelectrode was in contact to the perfusion catheter and connected to ahigh-impedance voltmeter (IsoMilivolt Meter; WPI). The referenceelectrode was grounded via a winged 21-gauge needle filled withisosmolar saline, and inserted subcutaneously in the abdomen. Measuringand reference electrodes consisted of Ag/AgCl with 3 M KCl agar bridges.

Tear secretion. To measure unstimulated tear production, phenol redthreads (Zone-Quick, Oasis Medical, Glendora, Calif., USA) were placedfor 10 s in the lateral canthi of isofluorane-anesthetized mice usingjewelers' forceps. Tear volume was measured as the length of threadwetting, as visualized under a dissecting microscope. Serialmeasurements were used to evaluate compound pharmacodynamics afterapplication of 2-□L drops of compound formulations (50-100 μM compoundin PBS containing 0.5% polysorbate and 0.5% DMSO) comparing to vehicle.

Lissamine green staining. To assess corneal epithelial disruption, 5 μLof lissamine green (LG) dye (1%) was applied to the ocular surface ofisofluorane-anesthetized mice. Photographs of the eye were taken using aNikon Digital camera adapted to an Olympus Zoom Stereo Microscope(Olympus, Center Valley, Pa., USA). Each corneal quadrant was scored ona 3-point scale by one blinded, trained observer, with the extent ofstaining in each quadrant classified as: 0, no staining; 1, sporadic(involving <25% of the total surface) staining; grade 2, diffusepunctate staining (25-75%); and grade 3, coalesced punctate staining(>75%). The total grade is reported as the sum of scores from all fourquadrants, ranging from 0 to 12.

Pharmacokinetics and tissue distribution. To determine the residencetime of CFTR activators in the pre-ocular mouse tear film, compoundswere recovered for liquid chromatography/mass spectroscopy (LC/MS)following single-dose ophthalmic delivery. Three eye washes (3 μL PBSeach) were recovered from the lateral and medial canthi with 5-μLmicrocapillary tubes (Drummond Scientific Co., Broomhall, Pa., USA)after manual eyelid blinking (9). Pooled washes were diluted withacetonitrile/water (1:1) containing 0.1% formic acid and analyzed byLC/MS using an Xterra MS C18 column (2.1 mm×100 mm, 3.5-μm particlesize) connected to a Waters 2695 HPLC solvent delivery system and aWaters Micromass ZQ mass spectrometer with positive electrosprayionization.

To study compound accumulation in systemic tissues, mouse blood, brain,kidney and liver were analyzed after 14 days of three-times dailytopical dosing (0.1 nmol, 2 μL, 50 μM). Blood samples were collectedfrom the left ventricle into K3 EDTA mini-tubes (Greiner, Kremsmunster,Austria) and centrifuged (28). The supernatant was extracted with anequal volume of ethyl acetate and the extract was dried with an airstream. Organs from treated and control mice were removed followingventricular perfusion with heparinized PBS (10 units/mL), weighed, mixedwith acetic acid and water (100 μL/g tissue), and homogenized (29).Ethyl acetate (10 mL/g tissue) was added, samples were vortexed andcentrifuged (3000 rpm for 15 min), and the ethyl acetate-containingsupernatant was evaporated. Residues obtained from organic extracts ofserum and organ homogenates were then reconstituted and analyzed byLC/MS as described above.

Mouse model of dry eye produced by lacrimal gland excision. A lacrimalgland excision (LGE) model of aqueous-deficient dry eye was adapted froma reported method (30). The extraorbital lacrimal gland was exposed oneach side of wild-type female BALB/c mice (7-8 weeks of age) by 3-mmlinear skin incisions. Lacrimal ducts were cauterized and the entiregland was removed bilaterally, avoiding facial vessels and nerves.Incisions were each closed with a single interrupted 6-0 silk suture.Orbital lacrimal tissue remained functional. Eyes with reduced cornealsensation (<5% of mice studied), as identified from neurotrophic cornealulcers within 1 day of LGE, were excluded. Mice were randomized toreceive either treatment (in both eyes) with CFTR_(act)-K089 (0.1 nmol)or vehicle. Mice were treated three times daily (8 AM, 2 PM and 8 PM)for 2 weeks starting on Day 1 after LGE. Tear secretion and LG stainingwere performed immediately prior to, and one hour after the initial doseon day 4, 10 and 14 after LGE.

Statistics. Data are expressed as the mean±standard error of the mean(SEM). For direct comparisons between two means, the two-sided Students't-test was used. For longitudinal measurements of tear secretion and LGscores in the dry eye prevention study, a linear mixed effectsregression was used, adjusting for non-independence of measurementstaken on the same eye and on both eyes of the same animal. Analysis wasconducted in R v.3.2 for Mac (R Foundation for Statistical Computing,Vienna, Austria), using packages lme4 and robustlmm.

Results.

Characterization of small-molecule CFTR activators. A cell-basedfunctional high-throughput screen of 120,000 compounds at 10 μMidentified 20 chemical classes of small-molecule activators of wild-typeCFTR that produced >95% of maximal CFTR activation. The screen was donein FRT epithelial cells co-expressing human wild-type CFTR and acytoplasmic YFP halide sensor in 96-well format (26, 31, 32). Secondaryscreening involved I_(sc) measurement in CFTR-expressing FRT cellspretreated with submaximal forskolin (50 nM). Twenty-one compounds fromeight chemical classes produced large increases in I_(sc) at 1μ□ (>75%of maximal current produced by 20 μM forskolin). A summary of EC₅₀ andV_(max) values for each compound is provided in FIG. 7.

Structures of activators from the four most active chemical classes areshown in FIG. 2A, along with corresponding concentration-dependence datafrom I_(sc) measurements. Each compound fully activated CFTR, as a highconcentration of forskolin produced little further increase in I_(sc),and the increase in I_(sc) was fully inhibited by a CFTR inhibitor,CFTR_(inh)-172. EC₅₀ values ranged from 20-350 nM (FIG. 2B). VX-770showed relatively weak activity against wild-type CFTR (FIG. 2C).CFTR_(act)-K032 and CFTR_(act)-K089 had lower potency and showed lessCFTR activation (˜50% V_(max)).

Compounds that directly target CFTR without causing elevation ofcellular cAMP were sought to minimize potential off-target effects (FIG.2D). Compounds producing elevations in intracellular cAMP (from ClassesO, Q, and R), probably by phosphodiesterase inhibition, were excludedfrom further consideration. Nanomolar-potency compounds from Classes B,J and K, which did not increase cAMP, were selected for furthercharacterization in living mice.

CFTR activators increase ocular surface chloride and fluid secretion invivo. An open-circuit potential difference (PD) method developed in ourlab was used to evaluate compound activity at the ocular surface invivo, as depicted in FIG. 3A (21). Cl⁻ channel function was quantifiedby measuring PD during continuous perfusion of the ocular surface with aseries of solutions that imposed a transepithelial Cl⁻ gradient andcontained various channel agonists and/or inhibitors. The ocular surfacewas first perfused with isosmolar saline to record the baseline PD.Amiloride was then added to the perfusate, followed by exchange to a lowCl⁻ solution in which Cl⁻ with an impermeant anion, gluconate. Thesemaneuvers allow for direct visualization of CFTR activation in responseto addition of candidate CFTR activators.

FIG. 3B shows large hyperpolarizations following exposure to CFTR-B074,CFTR_(act)-J027 and CFTR_(act)-K089, which were increased relativelylittle by forskolin and were reversed by CFTR_(inh)-172. In comparison,VX-770 produced minimal changes in ocular surface PD (FIG. 3C). FIG. 3Dsummarizes PD data for indicated activators, with data for additionalcompounds reported in FIG. 7. Control studies done in CF mice lackingfunctional CFTR showed no changes in PD following addition of each ofthe compounds tested, with a representative curve shown for CFTR-K032(FIG. 3E).

CFTR activators were next tested for their efficacy in augmenting tearproduction in mice. Preliminary experiments identified a standardophthalmic formulation (0.5% polysorbate) that increased compoundsolubility and duration-of-action. Following a single topical dose, theindirect CFTR activators cholera toxin, forskolin, and3-isobutyl-1-methylxanthine (IBMX) substantially increased basal tearsecretion at 30 min, but these effects were transient and undetectableafter 2 hours (FIG. 4A). However, the direct CFTR activators identifiedhere, CFTR_(act)-B074, CFTR_(act)-J027 and CFTR-K089, increased tearfluid secretion by approximately two-fold for at least four hours.VX-770 produced little tear secretion (FIG. 4B). Repeated topicaladministrations (three times daily for up to 2 weeks) produced sustainedtear hypersecretion without tachyphylaxis (FIG. 4C). CFTR activators didnot increase tear fluid secretion in CF mice, demonstrating selectiveCFTR targeting (FIG. 4D).

Toxicity and pharmacokinetics. Tear collection methods were validated bydemonstrating reproducible recovery of tetramethylrhodamine dextran (3kDa) from the ocular surface up to six hours after instillation. Thepharmacokinetics of CFTR-K089 at the ocular surface was determined byLC/MS of recovered tear washes. Following instillation of 0.1 nmol ofCFTR-K089 (2 μL, 50 μM) to the ocular surface, 7.9±2.4 pmol and0.011±0.004 pmol were recovered at five min and six hours, respectively(FIG. 5A). The amount of CFTR-K089 required for 50% CFTR activation(EC₅₀˜250 nM) lies between the dashed lines, reflecting concentrationscalculated from the highest and lowest reported normal tear volumes inmice (33, 34). The quantity of CFTR-K089 recovered from tear fluidpredicts therapeutic levels for at least six hours. Tear fluidpharmacokinetics of CFTR_(act)-J027 could not be measured because theLC/MS sensitivity was low for this compound.

Following two weeks of three times per day dosing, the amounts ofCFTR-K089 and CFTR_(act)-J027 were below the limits of detection (˜10and ˜700 fmol, respectively) in mouse blood, brain, liver and kidney,indicating minimal systemic accumulation. The chronically treated miceshowed no signs of ocular toxicity, as assessed by slit-lamp evaluationfor conjunctival hyperemia, anterior chamber inflammation, and lensclarity. LG staining showed no corneal or conjunctival epithelialdisruption (FIG. 5B). The compounds also produced no appreciable invitro cytotoxicity in cell cultures at concentrations up to 100 μM (FIG.5C).

CFTR activator prevents dry eye in a lacrimal gland excision model inmice. On the basis of its favorable tear film pharmacokinetics,CFTR-K089 was selected for testing in a mouse model of aqueous-deficientdry eye produced by LGE. Following extraorbital LGE in BALB/c mice,CFTR_(act)-K089-treated mice (0.1 nmol, administered three times daily)maintained basal tear volume, whereas tear volume from vehicle-treatedmice was significantly reduced at all subsequent time-points (FIG. 6A),and for at least 30 days. Similar to what was reported in C₅₇/b16 mice(30), decreased lacrimation in vehicle-treated BALB/c mice wasassociated with progressive epithelial disruption from Day 0 to Day 14,shown pictorially (FIG. 6B top) and quantitatively (FIG. 6C).CFTR_(act)-K089 not only restored tear secretion in LGE mice butremarkably prevented ocular surface epithelial disruption at all timepoints (FIG. 6B). Vehicle-treated eyes developed diffuse, progressivecorneal epitheliopathy (LG score increase of 7.3±0.6 by Day 14), whereaseyes treated with CFTR_(act)-K089 had minimal LG staining at all timepoints (LG score change, −0.6±0.6).

Discussion

A goal of this study was to investigate the potential utility ofsmall-molecule activators of CFTR for dry eye therapy. After severalprior development failures, dry eye remains an unmet need in oculardisease. In dry eye disorders, tear film hyperosmolarity stimulatespro-inflammatory signaling, secretion of cytokines andmetalloproteinases, and disruption of corneal epithelial cell integrity(35-38). By minimizing tear film hyperosmolarity, CFTR activation ispredicted to prevent these downstream ocular surface changes.

We identified small-molecule CFTR activators by high-throughputscreening that produced sustained Cl⁻-driven aqueous fluid secretionacross the ocular surface by a mechanism involving direct CFTRactivation rather than upstream cAMP signaling. The rationale to choosecompounds that activate CFTR directly was to minimize potentialoff-target effects of generalized cAMP stimulation and to reduce thelikelihood of tachyphylaxis for compounds targeting signaling receptors.These compounds had low-nanomolar EC₅₀ for activation of human CFTR invitro and produced full activation at higher concentrations. LargeCFTR_(act)-dependent PD hyperpolarizations and tear hypersecretion weredemonstrated in mice. Substantial compound activities in mice and humanswill facilitate translation of data here to humans.

We found that CFTR_(act)-K089 restored tear secretion and preventedepithelial disruption in an experimental mouse model of lacrimalinsufficiency. CFTR activators may be particularly suited for disordersof the lacrimal gland, such as primary Sjögren's syndrome, bystimulating fluid transport across the intact corneal and conjunctivalepithelia. CFTR activators probably exert their major pro-secretoryeffect at the ocular surface, although there is indirect for CFTRexpression and function in lacrimal gland (39-42). Direct stimulation oflacrimal secretion is unlikely in the studies here because of minimalcompound penetration to lacrimal tissues following topical delivery, andthe demonstrated compound efficacy in a model of lacrimal insufficiency.At the ocular surface, the conjunctiva probably contributes the bulk offluid secretion given its much larger surface area compared to comea(43).

Alternative pro-secretory therapies targeting different ocular surfaceion channels have been considered. The only FDA-approved CFTR activator,VX-770, was developed as a “potentiator” to treat CF by correcting thechannel gating of certain CFTR mutations (44). However, VX-770 showedrelatively little activity against wild-type CFTR in cell cultures andin mice in vivo. Chronic application of VX-770 may also diminish CFTRfunctional expression (24) and cause cataracts (seen in juvenile rats;ref. 42), which is likely an off-target effect because CFTR is notexpressed in lens.

CFTR_(act)-K089 and CFTR_(act)-J027 showed favorable pharmacodynamicsand could be conveniently administered topically several times daily ina standard ophthalmic formulation.

In conclusion, without wishing to be bound by theory, it is believedthat the efficacy of CFTR_(act)-K089 in a clinically relevant mousemodel of aqueous-deficient dry eye disease provides proof-of-principlefor topical, pro-secretory CFTR activator therapy to restore basal tearsecretion and prevent ocular surface pathology. Compared withimmunosuppressive approaches, CFTR activation has the advantage ofaddressing an early event in dry eye pathogenesis. Our data thus supportthe development potential of CFTR activators as first-in-class dry eyetherapy.

Referenced (Example 5)

[1]. The definition and definition of dry eye disease: report of theDefinition and Classification Subcommittee of the International Dry EyeWorkShop 2007 (DEWS) (2007). Ocul. Surf. 5, 65-204; [2]. Schaumberg, D.A., Dana, R., Buring, J. E., and Sullivan, D. A. (2009). Prevalence ofdry eye disease among US men: estimates from the Physicians' HealthStudies. Arch. Ophthalmol. 127, 763-768; [3]. Schaumberg, D. A.,Sullivan, D. A., Buring, J. E., and Dana, M. R. (2003). Prevalence ofdry eye syndrome among US women. Am. J. Ophthalmol. 136, 318-326; [4].Yu, J., Asche, C. V., and Fairchild, C. J. (2011). The economic burdenof dry eye disease in the United States: a decision tree analysis.Cornea 30, 379-387; [5]. Alves, M., Foseca, E. C., Alves, M. F., Malki,L. T., Arruda, G. V., Reinach, P. S., and Rocha, E. M. (2013). Dry eyedisease treatment: a systematic review of published trials and criticalappraisal of therapeutic strategies. Ocul. Surf. 11, 181-192; [6].Sheppard, J. D., Torkildsen, G. L., Lonsdale, J. D., D'Ambrosio Jr., F.A., McLaurin, E. B., Eiferman, R. A., Kennedy, K. S. and Semba, C. P.;OPUS-1 Study Group (2014). Lifitegrast ophthalmic solution 5.0% fortreatment of dry eye disease: results of the OPUS-1 phase 3 study.Ophthalmology 121, 475-483; [7]. Asbell P. A., and Spiegel S. (2010).Ophthalmologist perceptions regarding treatment of moderate-to-severedry eye: results of a physician survey. Eye Contact Lens 36, 33-38; [8].The epidemiology of dry eye disease: report of the EpidemiologySubcommittee of the International Dry Eye WorkShop 2007 (DEWS) (2007).Ocul. Surf. 5, 65-204; [9]. Thelin, W. R., Johnson, M. R., Hirsh, A. J.,Kublin, C. L. and Zoukhri, D. J. (2012). Effect of topically appliedepithelial sodium channel inhibitors on tear production in normal miceand in mice with induced aqueous tear deficiency. Ocul. Pharmacol. Ther.28, 433-438; [10]. Nichols, K. K., Yerxa, B. and Kellerman, D. J.(2004). Diquafosol tetrasodium: a novel dry eye therapy. Expert. Opin.Investig. Drugs 13, 47-54; [11]. Koh, S., Ikeda, C., Takai, Y.,Watanabe, H., Maeda, N., and Nishida, K. (2013). Long-term results oftreatment with diquafosol ophthalmic solution for aqueous-deficient dryeye. Jpn. J. Ophthalmol. 57, 440-446; [12]. Takamura, E., Tsubota, K,Watanabe, H., and Ohashi, Y.; Diquafosol Ophthalmic Solution Phase 3Study Group (2012). A randomised, double-masked comparison study ofdiquafosol versus sodium hyaluronate ophthalmic solutions in dry eyepatients. Br. J. Ophthalmol. 96, 1310-1315; [13]. Tauber, J., Davitt, W.F., Bokosky, J. E., Nichols, K K., Yerxa, B. R., Schaberg, A. E.,LaVange, L. M., Mills-Wilson, M. C., and Kellerman, D. J. (2004).Double-masked placebo-controlled safety and efficacy trial of diquafosoltetrasodium (INS365) ophthalmic solution for the treatment of dry eye.Comea 23, 784-792; [14]. Al-Nakkash, L., and Reinach, P. S. (2001).Activation of a CFTR-mediated chloride current in a rabbit cornealepithelial cell line. Invest. Ophthalmol. Vis. Sci. 42, 2353-2370; [15].Shiue, M. H., Gukasyan, H. J, Kim, K J., Loo, D. D., and Lee, V. H(2002). Characterization of cyclic AMP-regulated chloride conductance inthe pigmented rabbit conjunctival epithelial cells. Can. J. Physiol.Pharmacol. 80, 533-540; [16]. Turner, H. C., Bemstein, A., and Candia,O. A. (2002). Presence of CFTR in the conjunctival epithelium. Curr. EyeRes. 24, 182-187; [17]. Ansari, E. A., Sahni, K., Etherington, C.,Morton, A., Conway, S. P., Moya, E., and Littlewood, J. M. (1999).Ocular signs and symptoms and vitamin A status in patients with cysticfibrosis treated with daily vitamin A supplements. Br. J. Ophthalmol.83, 688-691; [18]. Botelho, S. Y., Goldstein, A M., and Rosenlund, M. L.(1973). Tear sodium, potassium, chloride, and calcium at various flowrates: children with cystic fibrosis and unaffected siblings with andwithout corneal staining. J. Pediatr. 83, 601-606; [19]. Morkeberg, J.C., Edmund, C., Prause, J. U., Lanng, S., Koch, C., and Michaelsen, K F.(1995). Ocular findings in cystic fibrosis patients receiving vitamin Asupplementation. Graefes Arch. Clin. Exp. Ophthalmol. 233, 709-713;[20]. Mrugacz, M., Kaczmarski, M., Bakunowicz-Lazarczyk, A., Zelazowska,B., Wysocka, J., and Minarowska, A. (2006). IL-8 and IFN-gamma in tearfluid of patients with cystic fibrosis. J. Interferon Cytokine Res. 26,71-75; [21]. Levin, M. H. and Verkman, A. S. (2005). CFTR-regulatedchloride transport at the ocular surface in living mice measured bypotential differences. Invest. Ophthalmol. Vis. Sci. 46, 1428-1434;[22]. Levin, M. H., Kim, J. K., Hu, J., and Verkman A. S. (2006).Potential difference measurements of ocular surface Na+ absorptionanalyzed using an electrokinetic model. Invest. Ophthalmol. Vis. Sci.47, 306-316; [23]. Yu, D., Thelin, W. R., Rogers, T. D., Stutts, M. J.,Randell, S. H., Grubb, B. R., and Boucher, R. C. (2012). Regionaldifferences in rat conjunctival ion transport activities. Am. J.Physiol. Cell Physiol. 303, C767-780; [24]. Cholon, D. M., Quinney, N.L., Fulcher, M. L., Esther Jr., C. R., Das, J., Dokholyan, N. V.,Randell, S. H., Boucher, R. C., and Gentzsch, M. (2014). Potentiatorivacaftor abrogates pharmacological correction of ΔF508 CFTR in cysticfibrosis. Sci. Transl. Med. 6, 246-296; [25]. Ramsey, B. W., Davies, J.,McElvaney, N. G., Tullis, E., Bell, S. C., Dřevínek, P., Griese, M.,McKone, E. F., Wainwright, C. E., Konstan, M. W., Moss, R., Ratjen, F.,Sermet-Gaudelus, I., Rowe, S. M., Dong, Q., Rodriguez, S., Yen, K.,Ordoñez, C., and Elbom, J. S.; VX08-770-102 Study Group (2011). A CFTRpotentiator in patients with cystic fibrosis and the G551D mutation. N.Engl. J. Med. 365, 1663-1672; [26]. Ma, T., Vetrivel, L., Yang, H.,Pedemonte, N., Zegarra-Moran, O., Galietta, L. J., and Verkman, A. S.(2002). High-affinity activators of CFTR chloride conductance identifiedby high-throughput screening. J. Biol. Chem. 277, 37235-37241; [27].Galietta, L. J., Springsteel, M. F., Eda, M., Niedzinski, E. J., By, K.,Haddadin, M. J., Kurth, M. J., Nantz, M. H., and Verkman, A. S. (2001).Novel CFTR chloride channel activators identified by screening ofcombinatorial libraries based on flavone and benzoquinolizinium leadcompounds. J. Biol. Chem. 276, 19723-19728; [28]. Esteva-Font, C., Cil,O., Phuan, P. W., Tao, S., Lee, S., Anderson, M. O., and Verkman, A. S.(2014) Diuresis and reduced urinary osmolality in rats produced bysmall-molecule UT-A-selective urea transport inhibitors. FASEB J. 28,3878-3890; [29]. Yao C, Anderson, M. O., Zhang J., Yang B., Phuan P. W.,and Verkman, A. S. (2012) Triazolothienopyrimidine inhibitors of ureatransporter UT-B reduce urine concentration. J. Am. Soc. Nephrol. 23,1210-1220; [30]. Stevenson, W., Chen, Y., Lee, S. M., Lee, H. S., Hua,J., Dohlman, T., Shiang, T., and Dana, R. (2014). Extraorbital lacrimalgland excision: a reproducible model of severe aqueous tear-deficientdry eye disease. Comea 33, 1336-1341; [31]\. Galietta, L. J., Haggie, P.M., and Verkman, A. S. (2001). Green fluorescent protein-based halideindicators with improved chloride and iodide affinities. FEBS Lett. 499,220-224; [32]. Galietta, L. V., Jayaraman, S., and Verkman, A. S.(2001). Cell-based assay for high-throughput quantitative screening ofCFTR chloride transport agonists. Am. J. Physiol. Cell Physiol. 281,C₁₇₃₄-1742; [33]. Sullivan, D. A., Krenzer, K. L., Sullivan, B. D.,Tolls, D. B., Toda, I., and Dana, M. R. (1999). Does androgeninsufficiency cause lacrimal gland inflammation and aqueous teardeficiency? Invest. Ophthalmol. Vis. Sci. 40, 1261-1265; [34].Villareal, A. L., Farley, W., and Pflugfelder, S. C. (2006). Effect oftopical ophthalmic epinastine and olopatadine on tear volume in mice.Eye Contact Lens 32, 272-276; [35]. Lemp, M. A., Bron, A., Baudouin, C.,Benitez Del Castillo, J., Geffen, D., Tauber, J., Foulks, G., Pepose, J.and Sullivan, B. D. (2011). Tear osmolarity in the diagnosis andmanagement of dry eye disease. Am. J. Ophthalmol. 151, 792-798; [36].Luo, L., Li, D. Q., Corrales, R. M., and Pflugfelder, S. C. (2005).Hyperosmolar saline is a proinflammatory stress on the mouse ocularsurface. Eye Contact Lens 31, 186-193; [37]. Liu, H., Begley, C., Chen,M., Bradley, A., Bonanno, J., McNamara, N., Nelson, J., and Simpson, T.(2009). A link between tear instability and hyperosmolarity in dry eye.Invest. Ophthalmol. Vis. Sci. 50, 3671-3679; [38]. Gilbard, J. P.,Carter, J., Sang, D., Refojo, M., Hanninen, L. and Kenyon, K. R. (1984).Morphologic effect of hyperosmolarity on rabbit corneal epithelium.Ophthalmology 91, 1205-1212; [39]. Rosemary, R., Evans, M., Cuthbert,A., MacVinish, J. L., Foster, D., Anderson, J., and Colledge, W. H.(1993). Nature Genetics 4, 35-41; [40]. Lu, M. and Ding, C. (2012).CFTR-mediated Cl(−) transport in the acinar and duct cells of rabbitlacrimal gland. Curr. Eye Res. 37, 671-677; [41]. Nandoskar, P., Wang,Y., Wei, R., Liu, Y., Zhao, P., Lu, M., Huang, J., Thomas, P.,Trousdale, M., and Ding, C. (2012). Changes of chloride channels in thelacrimal glands of a rabbit model of Sjögren syndrome. Comea 31,273-279; [42]. Kalydeco [Product Monograph] Laval, Quebec: VertexPharmaceuticals (Canada) Inc.; 2012; [43]. Watsky, M. A., Jablonski, M.,and Edelhauser, H. F. (1988). Comparison of conjunctival and cornealsurface area in rabbit and human. Curr. Eye Res. 7, 483-486; [44]. VanGoor, F., Hadida, S., and Grootenhuis, P. D. J. (2008). Pharmacologicalrescue of mutant CFTR function for the treatment of cystic fibrosis.Top. Med. Chem. 3, 91-120; [45]. Wolosin, J. M., and Candia, O. A.(1987). Cl— secretagogues increase basolateral K+ conductance of frogcorneal epithelium. Am. J. Physiol. 253, C₅₅₅-560; [46]. Kompella, U.B., Kim, K. J., and Lee, V. H. (1993). Active chloride transport in thepigmented rabbit conjunctiva. Curr. Eye Res. 12, 1041-1048; [47].Turner, H. C., Alvarez, L. J., and Candia, O. A. (2000). CyclicAMP-dependent stimulation of basolateral K(+)conductance in the rabbitconjunctival epithelium. Exp. Eye Res. 70, 295-305.

Example 6 Nanomolar-Potency Aminophenyl-1,3,5-Triazine CFTR ChlorideChannel activators for pro-secretory therapy of dry eye diseases

Compound numbering and substituents numbering within Example 6 is withrespect to Example 6 only.

Abstract. Dry eye disorders are a significant health problem for whichlimited therapeutic options are available. CFTR (cystic fibrosistransmembrane conductance regulator) is a major pro-secretory chloridechannel at the ocular surface. We previously identified, byhigh-throughput screening, aminophenyl-1,3,5-triazine CFTR_(act)-K089(1) that activated CFTR with EC₅₀˜250 nM, which when delivered topicallyincreased tear fluid secretion in mice and showed efficacy in anexperimental dry eye model. Here, functional analysis of synthesizedaminophenyl-1,3,5-triazine analogs elucidated structure-activityrelationships for CFTR activation and identified substantially morepotent analogs than 1. The most potent compound, 12, fully activatedCFTR chloride conductance with EC₅₀˜30 nM, without causing cAMP orcalcium elevation. 12 was non-toxic, stable at 100 μM in an ophthalmicvehicle, and rapidly metabolized by hepatic microsomes, which supportsits topical use in dry eye disease. Analogs with greater metabolicstability were also identified for possible treatment of liver and lungdisorders. Single topical administration of 25 pmol 12 increased tearvolume in wildtype mice with sustained action for 8 hours, and waswithout effect in CFTR-deficient mice. Topically delivered 12 may beefficacious in human dry eye diseases.

Introduction.

CFTR (cystic fibrosis transmembrane conductance regulator) is acAMP-gated chloride channel with pro-secretory activity in the airways,gastrointestinal organs, testis and exocrine glands [1-4].Loss-of-function mutations in CFTR cause cystic fibrosis, andinappropriate activation of CFTR causes certain secretory diarrheas suchas cholera [5-7]. CFTR is an important drug target for which majoradvances have been made in the development of potentiators andcorrectors of mutant CFTRs in cystic fibrosis, with two drugs havingreceived FDA approval [8]. Activators of wildtype CFTR have potentialclinical indications for pro-secretory therapy of constipation and dryeye disorders, and possibly for disorders of the liver, pancreas andairways [9-12], and CFTR inhibitors are under development for certainsecretory diarrheas and polycystic kidney disease [13,14].

We previously identified, by high-throughput screening, small-moleculeactivators of wildtype CFTR that function by a direct activationmechanism [12-15]. One class of phenylquinoxalinone CFTR activatorsshowed efficacy following oral administration in experimental mousemodels of constipation [9,10]. A second class of CFTR activators, whichincludes the aminophenyl-1,3,5-triazine CFTR-K089 (compound 1, FIG.14A), activated ocular surface CFTR activity in mice and increased tearfluid secretion after topical delivery [12]. In a mouse model ofaqueous-deficient dry eye produced by lacrimal gland excision, topicaldelivery of 1 three times daily corrected defective tear fluid secretionand prevented ocular surface clinical signs. An ideal dry eyetherapeutic would produce sustained tear fluid secretion with once ortwice daily dosing.

Here, we report structure-activity relationship studies ofaminophenyl-1,3,5-triazine CFTR activators with a focus on dry eyeapplications. Dry eye is a heterogeneous group of disorders associatedwith reduced tear volume and tear fluid hyperosmolarity, resulting inocular surface inflammation, eye discomfort and visual disturbance. Dryeye is a major health problem in an aging population, with five millionpatients in the U.S. age 50 and over [16,17]. The only approved dry eyedrugs, topical cyclosporine and lifitegrast, act by an anti-inflammatorymechanism [18,19]. CFTR activators would be first-in-class dry eyetherapeutics that function by a pro-secretory mechanism. Synthesis andcharacterization of aminophenyl-1,3,5-triazines here produced potentiallead candidates with substantially improved potency and in vivopro-secretory efficacy compared to 1, as well as compounds with greatermetabolic stability for potential treatment of non-ocular conditions.

Chemistry.

General synthesis of aminophenyl-1,3,5-triazine analogs. The synthesisof aminophenyl-1,3,5-triazine analogs was accomplished by serialsubstitution of cyanuric chloride using fluorinated alkoxides, diethylamine, and anilines (Scheme 1). Substitution of cyanuric chloride with1,1,1,3,3,3-hexafluoroisopropanol or 2,2,3,3-tetrafluoro-1-propanolafforded 2 and 3, respectively. Amination of 2 and 3 using diethyl amineafforded intermediates 4 and 5. Refluxing of 4 and 5 with anilineafforded 1 and 7a, respectively. Refluxing m- or p-F, Cl, NO₂,CO₂H-substituted anilines with 4 and 5 affordedaminophenyl-1,3,5-triazines 6a-g and 7b-g, respectively. 5 was refluxedwith 6-aminobenzothiazole, 6-aminobenzoxazole, 5- or 6-aminoindazoleunder basic condition to afford 6h-6k.

N-Methylamine-1,3,5-triazine analogs 11 and 12 were synthesized as shownin Scheme 2. Methylamine was substituted with cyanuric chloride toafford N-methyl-4,6-dichloro-1,3,5-triazine-2-amine 8 in 72% yield. With8 in hand, substitution with 1,1,1,3,3,3-hexafluoro-2-propanol or2,2,3,3-tetrafluoro-1-propanol afforded 9 and 10, respectively. Anilinesubstitution on 9 and 10 affordedN-methylamino-N′-phenylamino-1,3,5-triazine analogs 11 and 12,respectively.

General Synthesis Procedures. All solvents and chemicals were used aspurchased without further purification. The progress of all reactionswas monitored on Merck pre-coated silica gel plates (with fluorescenceat 254 nm) using ethyl acetate/n-hexane as solvent system. Columnchromatography was done with Fluka silica gel 60 (230-400 mesh ASTM)with specified solvent mixtures. Proton (¹H) and carbon (¹³C) NMRspectra were recorded on a Bruker Avance 300 or 500 (500.13 MHz for ¹H;125.76 MHz for ¹³C) using CDCl₃ (δ 7.26), CD₃OD (δ=4.87 and 3.31),acetone-d₆ (δ 2.05), or DMSO-d₆ (δ 2.5) as solvents. Chemical shifts aregiven in parts per million (ppm) (6 relative to residual solvent peakfor ¹H and ¹³C). HRMS was performed using a hybrid quadrupole orbitrapmass analyzer, QExactive (Thermo, Bremen, Germany), with an electrosprayionization source. The mass resolution was set as 70,000 at m/z 200 andthe mass accuracy was less than 3 ppm. Purity of all final compounds was95% or higher.

N,N-Diethyl-N′-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(6a). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 6a asa white powder (100 mg, 38%). ¹H NMR (300 MHz, CDCl₃): δ=7.60 (d, 2H,J=8.4 Hz), 7.34 (t, 2H, J=7.4 Hz), 7.08 (t, 1H, J=7.3 Hz), 6.06 (tt, 1H,J=4.9, 53.0 Hz), 3.68-3.59 (m, 2H), 4.78-4.70 (m, 4H), 1.28-1.20 (m,6H); ¹³C NMR (75 MHz, CDCl₃): δ=169.2, 165.2, 164.8, 138.6, 128.7,123.1, 120.0, 114.4 (t, J=26 Hz), 112.3 (t, J=34 Hz), 109.0 (t, J=34Hz), 105.7 (t, J=34 Hz), 62.1 (t, J=30 Hz), 42.1, 41.7, 13.1, 12.9; HRMS(ESI): m/z calculated for C16H20F4N5O [M+H+]: 374.1599. Found: 374.1597.

3-{[4-(Diethylamino)-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazin-2-yl]amino}benzoicacid (6b). Purified by flash chromatography (1:5 EtOAc/Hex) to afford 6bas a white powder (60%). ¹H NMR (300 MHz, acetone-d6): δ=8.68 (brs, 1H),7.92 (m, 1H), 7.74-7.71 (m, 1H), 7.46 (t, 1H, J=7.84 Hz), 6.45 (tt, 1H,J=Hz), 4.91-4.82 (m, 2H), 3.74-3.65 (m, 4H), 1.28-1.18 (m, 6H); ¹³C NMR(75 MHz, acetone-d6): δ=169.12, 164.68, 159.89, 157.96, 142.33, 134.51,131.28, 125.52, 121.99, 118.77, 115.45, 115.27, 114.35, 112.35, 111.29,111.01, 110.73, 110.07, 109.29, 109.02, 108.75, 107.31, 107.03, 106.76,62.71, 62.37, 62.13, 61.89, 42.13, 41.84, 29.67, 13.06, 12.86, 12.77;HRMS (ESI): m/z calculated for C17H20F4N5O3 [M+H+]: 418.1497. Found:418.1501.

N,N-Diethyl-N-(3-fluorophenyl)-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(6c). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 6c asa white powder (40%). ¹H NMR (300 MHz, CDCl₃): δ=7.73 (dt, 1H, J=2.1,7.73 Hz), 7.37 (brs, 1H), 7.26 (td, 1H, J=6.31, 8.19 Hz), 7.12 (ddd, 1H,J=0.9, 2.0, 8.16 Hz), 6.76 (ddt, 1H, J=0.9, 2.5, 8.2 Hz), 6.02 (tt, 1H,J=4.92, 53.01 Hz), 4.74 (tt, 2H, J=1.6, 12.4 Hz), 3.64 (sex, 4H, J=7.02Hz), 1.23 (n, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=169.2, 165.2, 164.8,164.6, 161.4, 140.4, 140.3, 129.8, 129.7, 114.9, 114.9, 114.7, 114.3,114.0, 112.7, 112.3, 111.0, 109.7, 109.47, 109.42, 109.0, 108.5, 107.3,107.0, 105.7, 62.5, 62.1, 61.7, 42.3, 41.9, 13.0, 12.8; HRMS (ESI): m/zcalculated for C16H19F5N5O [M+H+]: 392.1505. Found: 392.1505.

N-(3-Chlorophenyl)-N,N-diethyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(6d). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 6d asa white powder (81%). ¹H NMR (300 MHz, CDCl₃): δ=7.99 (brs, 1H), 7.25(m, 3H), 7.04 (dt, 1H, J=7.01, 2.01 Hz), 6.02 (t, 1H, J=4.89 Hz), 4.73(t, 2H, J=12.3 Hz), 3.64 (m, 4H), 1.84 (brs, 1H), 1.25 (n, 6H); ¹³C NMR(75 MHz, CDCl₃): δ=169.2, 165.2, 164.7, 139.9, 134.5, 129.6, 122.9,120.0, 117.6, 114.5, 114.3, 114.1, 112.3, 111.3, 111.0, 110.7, 109.3,109.0, 108.8, 107.3, 107.0, 106.8, 62.4, 62.1, 61.9, 42.4, 42.0, 13.0,12.9; HRMS (ESI): m/z calculated for C16H19ClF4N40 [M+H+]: 408.1209.Found: 408.1210.

N,N-Diethyl-N-(3-nitrophenyl)-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(6e). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 6e asa yellowish powder (61%). ¹H NMR (300 MHz, CDCl₃): δ=9.12 (brs, 1H),8.15 (brs, 1H), 7.90 (dd, 1H, J=8.20, 1.58 Hz), 7.57 (d, 1H, J=7.57 Hz),7.44 (t, 1H, J=8.04 Hz), 5.96 (m, 1H), 4.75 (t, 2H, J=12.61 Hz), 3.72(q, 2H, J=6.94 Hz), 3.65 (q, 2H, J=6.94), 2.12 (brs, 1H), 1.31 (t, 3H,J=7.09 Hz), 1.24 (t, 3H, J=7.09 Hz); ¹³C NMR (75 MHz, CDCl₃): δ=169.1,164.9, 164.7, 148.6, 140.1, 129.2, 124.8, 117.4, 116.2, 114.6, 114.5,114.2, 114.0, 112.3, 111.3, 111.0, 110.7, 109.3, 109.0, 108.7, 107.3,107.0, 106.7, 62.3, 62.1, 61.8, 42.6, 42.1, 12.9, 12.8; HRMS (ESI): m/zcalculated for C16H19F4N6O3 [M+H+]: 419.1450. Found: 419.1449.

4-({4-(Diethylamino)-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazin-2-yl}amino)benzoicacid (6f). Purified by flash chromatography (1:10 EtOAc/Hex) to afford6f as a white powder (37%). ¹H NMR (300 MHz, acetone-d6): δ=8.97 (brs,1H), 8.04-7.96 (m, 4H), 6.45 (tt, 1H, J=5.07, 52.41 Hz), 4.88 (t, 2H,J=13.5 Hz), 3.74-3.64 (m, 4H), 1.29-1.19 (m, 6H); ¹³C NMR (75 MHz,acetone-d6): δ=169.7, 166.4, 165.5, 165.4, 144.2, 130.5, 124.0, 118.8,109.6, 109.1, 62.1, 61.7, 61.4 (t, J=Hz), 41.8, 41.5, 12.4, 12.3; HRMS(ESI): m/z calculated for C₁₇H20F4N503 [M+H+]: 418.1497. Found:418.1499.

N,N-Diethyl-N-(4-fluorophenyl)-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(6g). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 6g asa white powder (45%). ¹H NMR (300 MHz, CDCl₃): δ=7.53 (m, 2H), 7.36(brs, 1H), 7.02 (m, 2H), 6.03 (m, 1H), 4.73 (m, 2H), 3.61 (m, 4H), 1.24(m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=169.1, 164.6, 159.8, 157.9, 142.3,134.5, 131.2, 125.5, 121.9, 118.7, 115.4, 115.2, 114.3, 112.3, 111.2,111.0, 110.7, 110.07, 109.2, 109.0, 108.7, 107.3, 107.0, 106.7, 62.7,62.3, 62.1, 61.8, 42.1, 41.8, 29.6, 13.0, 12.8, 12.7; HRMS (ESI): m/zcalculated for C16H19F5N50 [M+H+]: 392.1505. Found: 392.1503.

3-({4-(diethylamino)-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazin-2-yl}amino)benzoicacid (7a). Purified by flash chromatography (1:6 EtOAc/Hex) to afford 7aas a white powder (55%). ¹H NMR (300 MHz, acetone-d6): δ=9.02 (brs, 1H),8.79 (brs, 1H), 7.90 (d, 1H, J=8.07 Hz), 7.76 (dt, 1H, J=1.29, 7.86 Hz),7.48 (t, 1H, J=7.92 Hz), 6.79 (quint, 1H, J=6.4 Hz), 3.78-3.66 (m, 4H),1.29-1.20 (m, 6H); ¹³C NMR (75 MHz, acetone-d6): δ=168.6, 166.8, 165.3,139.6, 131.1, 128.7, 123.9, 123.1, 121.2, 119.3, 68.9, 68.5, 68.0, 67.6,67.1 (quint, J=34 Hz), 42.1, 41.6, 12.35, 12.32; HRMS (ESI): m/zcalculated for C17H18F6N5O3 [M+H+]: 454.1309. Found: 454.1311.

N,N-Diethyl-N-(3-fluorophenyl)-6-[(1,1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine-2,4-diamine(7b). Purified by flash chromatography (1:8 EtOAc/Hex) to afford 7b as awhite powder (45%). ¹H NMR (300 MHz, CDCl₃): δ=7.69 (m, 1H), 7.48 (brs,1H), 7.26 (m, 1H), 7.11 (d, 1H, J=7.88 Hz), 6.79 (td, 1H, J=8.2, 2.5Hz), 6.31 (dt, 1H, J=12.37, 6.27 Hz), 3.64 (m, 4H), 1.25 (m, 6H); ¹³CNMR (75 MHz, CDCl₃): δ=34168.5, 165.1, 164.8, 164.6, 161.3, 140.1,139.9, 129.8, 129.7, 122.7, 118.9, 115.1, 110.0, 109.7, 107.6, 107.2,68.8, 68.4, 67.9 (t, J=34 Hz), 42.6, 42.2, 12.8, 12.7; HRMS (ESI): m/zcalculated for C16H17F7N5O [M+H+]: 428.1316. Found: 428.1316.

N,N-Diethyl-6-[(1,1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N-(3-nitrophenyl)-1,3,5-triazine-2,4-diamine(7d). Yellowish powder, 62% yield. ¹H NMR (300 MHz, CDCl₃): δ=9.15 (brs,1H), 8.23-7.74 (m, 2H), 7.70-7.34 (1 m, 2H), 6.31 (quint, 1H, J=6.15Hz), 3.74 (q, 2H, J=6.94 Hz), 3.63 (q, 2H, J=7.04 Hz), 1.89 (brs, 1H,1.33 (t, 3H, J=7.09 Hz), 1.24 (t, 4H, J=7.09 Hz); ¹³C NMR (75 MHz,CDCl₃): δ=168.4, 165.1, 164.8, 148.6, 139.8, 129.3, 128.4, 126.3, 125.2,122.6, 118.8, 115.1 (q, J=280 Hz), 125.0, 117.8, 114.8, 68.9, 68.4, 68.0(t, J=34 Hz), 42.9, 42.4, 12.8, 12.7; HRMS (ESI): m/z calculated forC16H17F6N6O3 [M+H+]: 455.1261. Found: 455.1263.

4-({4-(Diethylamino)-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazin-2-yl}amino)benzoicacid (7e). Purified by flash chromatography (1:10 EtOAc/Hex) to afford7e as a white powder (56%). ¹H NMR (300 MHz, acetone-d6): δ=9.12 (brs,1H), 8.05-7.89 (m, 4H), 6.80 (quint, 1H, J=6.39 Hz), 3.72, (quint, 4H,J=7.2 Hz), 1.31-1.18 (n, 7H); ¹³C NMR (75 MHz, acetone-d6): δ=168.66,165.91, 165.44, 165.34, 137.51, 133.65, 132.32, 131.53, 126.93, 123.46,123.20, 123.16, 121.85, 119.46, 119.42, 115.73, 113.32, 112.69, 111.18,109.84, 68.94, 68.44, 68.01, 67.56, 42.06, 41.55, 12.45, 12.31; HRMS(ESI): m/z calculated for C17H18F6N5O3 [M+H+]: 454.1309. Found:454.1308.

N,N-Diethyl-N-(4-fluorophenyl)-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine-2,4-diamine(7f). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 7f asa white powder (35%). ¹H NMR (300 MHz, CDCl₃): δ=7.55-7.50 (m, 2H), 7.25(brs, 1H), 7.08-7.01 (m, 2H), 6.32 (quint, 1H, J=6.30 Hz), 3.61 (dq, 4H,J=3.06, 7.08 Hz), 1.23 (q, 6H, J=7.17 Hz); ¹³C NMR (75 MHz, CDCl₃):δ=168.5, 165.1, 160.7, 157.4, 134.2, 122.7, 122.2, 118.9, 115.5, 115.2,69.2, 68.8, 68.3, 67.9 (quint, J=34 Hz), 42.3, 42.0, 12.9, 12.8; HRMS(ESI): m/z calculated for C16H17F7N50 [M+H+]: 428.1316. Found: 428.1319.

N,N-Diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine(7g). Purified by flash chromatography (1:10 EtOAc/Hex) to afford 7g asa yellowish powder (75%). ¹H NMR (300 MHz, CDCl₃): δ=8.37 (brs, 1H),8.24-8.19 (m, 2H), 7.84-7.79 (m, 2H), 6.33 (quint, 1H, J=6.24 Hz),3.71-3.60 (m, 4H), 1.27 (dt, 6H, J=7.05, 17.33 Hz); ¹³C NMR (75 MHz,(CDCl₃): δ=168.4, 164.9, 164.8, 144.7, 142.7, 126.3, 122.6, 118.8, 115.1(q, J=281 Hz), 124.8, 119.2, 69.4, 68.9, 68.5, 68.0, 67.6 (quint, J=34Hz), 42.7, 42.4, 12.78, 12.75; HRMS (ESI): m/z calculated forC16H17F6N6O3 [M+H+]: 455.1261. Found: 455.1264.

N,N-Diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N′-(1H-indazol-5-yl)-1,3,5-triazine-2,4-diamine(7h). Purified by flash chromatography (1:6 EtOAc/Hex) to afford 7h as aoff-white powder (28%). ¹H NMR (300 MHz, acetone-d6): δ=12.22 (brs, 1H),8.85 (brs, 1H), 8.29 (brs, 1H), 8.03 (d, 1H, J=0.96 Hz), 7.67 (d, 1H,J=8.94 Hz), 7.57 (d, 1H, J=8.94 Hz), 6.79 (quint, 1H, J=6.45 Hz), 3.68(q, 4H, J=6.99 Hz), 1.28-1.17 (m, 6H); ¹³C NMR (75 MHz, acetone-d6):δ=168.6, 165.9, 165.4, 165.3, 137.5, 133.6, 132.3, 131.5, 126.9, 123.4,123.2, 123.1, 121.8, 119.46, 119.42, 115.7, 113.3, 112.6, 111.1, 109.8,68.4, 68.0, 67.5 (quint, J=34 Hz), 42.0, 41.5, 12.4, 12.3; HRMS (ESI):m/z calculated for C17H18F6N7O [M+H+]: 450.1472. Found: 450.1472.

N,N-Diethyl-6-[(1,1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N-(1H-indazol-6-yl)-1,3,5-triazine-2,4-diamine(7i). Purified by flash chromatography (1:6 EtOAc/Hex) to afford 7i as aoff-white powder (28%). ¹H NMR (300 MHz, acetone-d6): 5=8.96 (brs, 1H),8.38 (brs, 1H), 7.97 (d, 1H, J=1.05 Hz), 7.71 (dd, 1H, J=0.72, 8.67 Hz),7.34 (dd, 1H, J=1.77, 8.70 Hz), 6.79 (quint, 1H, J=6.48 Hz), 3.79 (m,4H), 1.32-1.21 (m, 6H); ¹³C NMR (75 MHz, acetone-d6): δ=168.6, 165.3,162.8, 140.9, 137.7, 133.5, 126.9, 123.1, 120.4, 119.5, 115.3, 68.0,42.1, 41.6, 12.4, 12.2; 169.12, 164.68, 159.89, 157.96, 142.33, 134.51,131.28, 125.52, 121.99, 118.77, 115.45, 115.27, 114.35, 112.35, 111.29,111.01, 110.73, 110.07, 109.29, 109.02, 108.75, 107.31, 107.03, 106.76,62.71, 62.37, 62.13, 61.89, 42.13, 41.84, 29.67, 13.06, 12.86, 12.77;HRMS (ESI): m/z calculated for C17H18F6N7O [M+H+]: 450.1472. Found:450.1475.

N-(1,3-Benzoxazol-6-yl)-N,N-diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine-2,4-diamine(7j). Purified by flash chromatography (1:8 EtOAc/Hex) to afford 7j as apurple powder (25%). ¹H NMR (300 MHz, acetone-d6): δ=9.08 (brs, 1H),8.46 (brs, 1H), 8.39 (s, 1H), 7.70 (d, 1H, J=8.61 Hz), 7.63 (dd, 1H,J=1.92, 8.61 Hz), 6.80 (quint. 1H, J=6.75 Hz), 3.76-3.67 (m, 4H),1.31-1.18 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=168.50, 164.53, 152.91,149.69, 136.08, 134.72, 123.52, 121.96, 119.88, 119.68, 112.77, 97.34,68.99, 68.70, 68.45, 42.71, 42.36, 12.92, 12.85; HRMS (ESI): m/zcalculated for C17H17F6N6O2 [M+H+]: 451.1312. Found: 451.1313.

N-(1,3-Benzothiazol-6-yl)-N,N-diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine-2,4-diamine(7k). Purified by flash chromatography (1:7 EtOAc/Hex) to afford 7k as aoff-white powder (40%). ¹H NMR (300 MHz, CDCl₃): δ=8.93 (brs, 1H), 8.47(brs, 1H), 8.02 (d, 1H, J=8.51 Hz), 7.52 (dd, 2H, J=8.83, 2.21 Hz), 6.30(m, 1H), 3.66 (m, 4H), 1.27 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=168.5,164.5, 152.9, 149.6, 136.0, 134.7, 123.5, 121.9, 119.8, 119.6, 112.7,97.3, 68.9, 68.7, 68.4, 42.7, 42.3, 12.9, 12.8; HRMS (ESI): m/zcalculated for C17H17F6N60S [M+H+]: 467.1083. Found: 467.1089.

Results

Preliminary structure-activity analysis of commercially availableanalogs. Initial evaluation of compound activity was done using acell-based plate reader assay of CFTR function. FIG. 14B summarizespreliminary SAR analysis from functional assays on 91 commerciallyavailable analogs of 1 (data for all commercial compounds provided inTable 2). The commercial 1,3,5-triazine analogs tested here weretri-substituted at the 2-, 4-, 6-positions with various substituents,including alkoxide, aniline (amino-phenyl), and primary or secondaryamines. SAR analysis revealed that trisubstitued-1,3,5-triazines whosethree substituents contain a fluorinated alkoxide (R¹), an aniline (R²)and an alkylamine (R³) gave the best potency (compounds K032, K059 andK089, Table 2). Analogs with two-alkoxide substituents ornon-fluorinated alkoxide greatly reduced activity (K010-018, K023).Further, ortho-substituted aniline (R²) reduced activity as compared tometa- and para-substitution (K007, K032, K059). Electron-donating groupsincluding methoxy, acetyl, trifluoromethoxy (K001, K024-028, K079)reduced activity as compared to electron-withdrawing group such as nitroas in K032. Finally, sterically bulky alkyamines (R³) including cyclic,tert-butyl- and isobutylamine reduced activity (K029, K056, K060-065).Short alkylamines such as methylamine and diethylamine gave greatestactivity.

TABLE 2 Chemical structures and data for CFTR activation. % Activa- tion(EC₅₀, Code structure nM) K001

32 K002

63 K003

16 K004

16 K005

29 K006

16 K007

21 K008

32 K009

57 K010

21 K011

20 K012

19 K013

15 K014

18 K015

 6 K016

19 K017

16 K018

60 K019

28 K020

37 K021

16 K022

53 K023

41 K024

64 K025

19 K026

16 K027

20 K028

23 K029

 9 K030

16 K031

61 K032

105  (70) K033

22 K034

73 K035

55 K036

46 K037

36 K038

53 K039

27 K040

17 K041

35 K042

15 K043

19 K044

55 K045

15 K046

16 K047

17 K048

46 K049

28 K050

66 K051

45 K052

15 K053

24 K054

14 K055

42 K056

38 K057

28 K058

37 K059

90 (750)  K060

38 K061

31 K062

49 K063

41 K064

40 K065

38 K066

87 (2900)  K067

49 K068

84 (4100)  K069

37 K070

28 K071

25 K072

31 K073

19 K074

25 K075

32 K076

46 K077

29 K078

28 K079

40 K080

26 K081

24 K082

28 K083

54 K084

22 K085

25 K086

20 K087

47 K088

33 K089

111  (250)  K090

52 K091

43 % Activation of commercial analogs. For compounds >80% activationwere measured EC₅₀s from full-dose response assay.

Because the initial SAR data from commercial analogs was limited andconfounded by simultaneous variations in R¹, R² and R³, we synthesizedtargeted trisubstituted-1,3,5-triazine analogs. EC₅₀ values for CFTRactivation for the synthesized compounds are summarized in Tables 3-5.Active compounds produced full activation of CFTR, as seen by thesimilar activity produced by maximal forskolin (20 μM), but withdifferent EC₅₀ values.

Modifications of substituted anilines and fluorinated alkoxides. Sinceboth fluorinated alkoxide (R′) and short alkylamine (R³) appeared to becrucial for activity, we first prepared N-aryl-1,3,5-triazine amineswith R³ as diethylamine and explored the activity of two fluorinatedalkoxides, 1,1,1,3,3,3-hexafluoro-2-propanol and2,2,3,3-tetrafluoro-1-propanol. As shown in Table 3,hexafluoroisopropanol (1) was more potent than tetrafluoropropanol (7a).With various aryl (R²) substituent, hexafluoroisopropanol analogs(6a-6f) were ˜3 to 8-fold more potent than correspondingtetrafluoropropanol analogs (7b-7g). To investigate aniline substituents(R²), meta- and para-substituted aniline analogs with electron-neutraland electron-withdrawing groups were prepared. In general,meta-substituted anilines (6a and 6b) were more active thanpara-substituted aniline analogs (6e and 6f). Among meta-substitutedanilines, F— (6b), NO₂-(6d), and CO₂H-aniline (6a) had comparable orslightly better potency than non-substituted aniline (1), butchloroaniline (6c) had significantly reduced activity. With this resultwe hypothesized that hydrogen bonding formation is important to theactivity, so we kept focus on the aniline moiety (R²) and synthesizedmore analogs on R².

TABLE 3 Structures of N,N-diethyl-N′-aryl-1,3,5-triazine diamine analogsand their EC₅₀ (μM).

R¹ = OCH(CF₃)₂ 6a 6b 6c 6d  0.16  0.14 2.8 0.2 R¹ = OCH₂(CF₂)₂H 7b 7c 7d7e 0.5 1.6 5.4 1.2

R¹ = OCH(CF₃)₂ 6e 6f 6g 1 0.9 0.7 18  0.24 R¹ = OCH₂(CF₂)₂H 7f 7g 7a 2.54.8 2.0

Modifications of bicyclic anilines. To further investigate anilinesubstituents, compounds were synthesized with bicyclic anilines at theR² position with diethylamine (R³) and hexafluoroisopropanol (R¹),including 5- or 6-aminoindazole (6h and 6i), 6-aminobenzoxazole (6j) and6-aminobenzothiazole (6k). As shown in Table 4, the indazole analog 6hwas ˜3-fold more potent than its regioisomer 6i, which also supports theconclusion that hydrogen bonding formation on meta-position increasesactivity. Replacement of indazole (6h) by benzoxazole (6j) were morepotent than 1. Notably, replacing benzoxazole (6j) by benzothiazole (6k)increased potency by ˜3-fold to EC₅₀˜50 nM, supporting the importance ofhydrogen bonding acceptor function.

TABLE 4 Substitution with bicyclic anilines and their activity.

Modifications with N-methylamine. Finally, N,N-diethylamine at R³ wasreplaced by methylamine with two fluorinated alkoxides (Table 5).Notably, N-methylamine-2,2,3,3-tetrafluoro-1-propanoxy-1,3,5-triazine 12had EC₅₀˜50-fold better than diethylamine analog 6a. Interestingly, fordiethylamine (R³) analogs, hexafluoropropanol (1) is more potent thantetrafluoropropanol (7a), whereas for methylamine (R³) analogs,tetrafluoropropanol analog 12 was ˜3 fold more potent thanhexafluoropropanol analog 11. The most potent compounds emerging fromthe SAR analysis were chosen for biological characterizations andefficacy studies.

TABLE 5 N-methyl-N′-phenyl-1,3,5-triazine diamine analogs and theiractivity.

Biology

In vitro characterization of amino-phenyl-1,3-5-triazines. Short-circuitcurrent measurements were done on CFTR-expressing FRT cells using atransepithelial chloride gradient and following permeabilization of thecell basolateral membrane, such that short-circuit current magnitudeprovides a quantitative, linear measure of CFTR chloride conductance.Representative data in FIG. 15A for 6k and 12 shows a small increase incurrent with addition of a low concentration of forskolin, followed byconcentration-dependent increases in current following compoundadditions. The compounds produced full CFTR activation, as littlefurther increase in current was seen following a maximal concentrationof forskolin. EC₅₀ values were 30 nM for 6k and 31 nM for 12 (FIG. 15B).

Possible off-target effects on cell signaling and relevant non-CFTR ionchannels were investigated. 6k and 12 at 10 μM did not elevate cellularcAMP (FIG. 16A), increase cytoplasmic calcium, or inhibit theATP-stimulated elevation in cytoplasmic calcium (FIG. 16B). Also, 6k and12 at 10 μM neither inhibited nor activated calcium-activated chloridechannels in TMEM16A-expressing FRT cells (FIG. 16C) or HT-29 cells (FIG.16D). To further investigate specificity, the major ion transportpathways in human bronchial epithelial (HBE) cells were studied byshort-circuit current. HBE cells were sequentially treated withamiloride to inhibit the epithelial sodium channel (ENaC), forskolin toactivate CFTR, CFTR_(inh)-172 to inhibit CFTR, and ATP to activate CaCC,which produced the anticipated changes in short-circuit current incontrol cells (FIG. 16E left). Pretreatment of cells for 10 min with 10μM 6k or 12 did not alter short-circuit current responses to ENaC,maximal forskolin or ATP, indicating that the compound do not effectENaC or CaCC, or the supporting epithelial cell transporters necessaryto generate current (NKCC1, epithelial K⁺ channels, Na+/K⁺ pump). 12 didnot activate CFTR in HBE cells in the absence of basal CFTRphosphorylation produced by forskolin (FIG. 16E, right top), whereas 0.1μM 12 fully activated CFTR following 100 nM forskolin (FIG. 16E, rightbottom).

Pharmacological Properties and In Vivo Efficacy.

Compounds were further studied for their solution and metabolicstability, and cellular toxicity. FIG. 17A shows that 6k and 12 did notproduce significant toxicity using an Alamar blue assay, with 33% DMSOas positive control. When dissolved at 100 μM in an ophthalmic vehiclecontaining 0.3% CMC in Ringer's solution, 12 remained chemically stablefor 24 h at 40° C. as assayed by LC/MS (data not shown). To assessmetabolic stability, compounds at 5 μM were incubated with rat hepaticmicrosomes in the presence of NADPH. FIG. 17B shows the kinetics ofcompound metabolism, and LC/MS profiles of 12. 1 and 12, which containnon-substituted anilines, were metabolized rapidly with <40% of theoriginal compounds remaining at 15 min, whereas 6j and 6k, which containbicyclic anilines, were metabolized slowly with ˜60% of the originalcompounds remaining at 60 min. The benzoxazole-containing analog 6jshowed greater metabolic stability than benzothiazole analog 6k, whichmay be due to relative lability of the sulfide in benzothiazole versusthe oxygen in benzoxazole. Low metabolic stability is desirable for adry eye therapeutic to minimize systemic exposure following potentialabsorption, whereas high metabolic stability is desirable for liver andlung application of CFTR activators where systemic exposure and organaccumulation are needed.

Measurements of tear fluid volume were done following a singleophthalmic dosing of test compound in 2.5 μL of an ophthalmic vehicle,or vehicle alone as control. Compound 12 was selected followingpreliminary testing of compound efficacy at 6 hours. At 100 μM (250pmol) 12 produced a sustained increase in tear volume for 8 hours thatreturned to baseline over 12-24 h, with improved kinetics compared to 1(FIG. 18A). As evidence for compound specificity, 12 did not increasetear volume in CF mice, which lack functional CFTR (FIG. 18B). Adose-response study was done to determine the minimal amount of 12 thatproduced sustained tear fluid secretion. FIG. 18C shows similar activityof 12 at doses down to 25 pmol with maximal tear volume at 1-3 hours andsustained effect to 8 hours.

Discussion

The structure-activity study here identified analogs of 1 withsubstantially greater CFTR activation potency, with the most potentcompound 12 having EC₅₀˜30 nM, CFTR selectivity, and sustained in vivoefficacy for at least 8 hours in mice. CFTR selectivity was demonstratedin cellular studies and by the lack of increase in tear fluid volume inCFTR-deficient mice. 12 was chemically stable, including when formulatedin a standard ophthalmic vehicle. Minimal systemic exposure of 12 ispredicted based on the small amount needed to stimulate tear secretionand its rapid predicted hepatic metabolism. Further studies supportingits clinical development will be needed, such as ocular tissuepharmacokinetics, long-term toxicity and patient tolerability, which arebeyond the scope of this medicinal chemistry study. Of note, synthesizedanalogs 6j and 6k had substantially greater in vitro metabolic stabilitythan 1 or 12, and therefore have potential utility in therapy of liverand lung diseases where systemic exposure is necessary.

Various biological activities have been reported for the 1,3,5-triazinesscaffold. Atrazine, a chlorotriazine with two alkylamines substituent,is a widely used herbicide²⁰. SomeN,N-di-aminobenzyl-N-isopropyl-1,3,5-triazines were found to haveanti-tubulin activities in vitro and in vivo²¹. In addition, triazinescontaining bulky piperazinyl- and cyclohexylamino-substitutions werereported to have cathepsin inhibition activity [22]. The substituents inthese biologically active triazines are very different from those thatconferred CFTR activation in this study. Structure-activity analysis ofcommercial and synthesized aminophenyl-1,3,5-triazines determined thatthree specific substituent—alkoxide, alkylamine, and phenylamine—on thetriazine are needed for CFTR activity. For each of the substituent, (i)fluorinated alkoxides showed superior activity than non-fluorinatedalkoxides; (ii) short and linear alkylamines are crucial for activitywhile bulky amines greatly reduced activity; and (iii) phenylaminescontaining electron-withdrawing groups, including nitro, carboxylic acidor fluoride, in the meta-position increased activity. Of note, compound12 has drug-like properties, including favorable molecular weight (331Da), topological polar surface area (70.3 Å²) and c Log P (4.51), thatlatter much improved compared to c Log P of 5.99 for 1.

CFTR is a compelling target for dry eye therapy as it is expressed incorneal and conjunctival epithelia at the ocular surface [23-26] whereit functions as a prosecretory chloride channel [27,28]. While CFTR islargely inactive under basal conditions, when activated it has thecapacity to drive secretion of tear fluid onto the ocular surface. Byanalogy, CFTR is largely inactive in the intestine under basalconditions, but when activated by bacterial enterotoxins as in cholerait can promote massive secretion of fluid into the intestinal lumen. Astopically delivered CFTR activators act primary on ocular surfaceepithelia, they are predicted to be efficacious in dry eye produced bymultiple etiologies, including disorders targeting lacrimal glands suchas Sjogren's syndrome. It is noted, however, that a pure prosecretorytherapeutic replaces primarily the aqueous compartment of the tear film,which also contains lipid and mucin components. This potentiallimitation also applies to anti-absorptive therapeutics in developmenttargeting the ENaC sodium channel [29]. Notwithstanding this potentiallimitation, the demonstration of efficacy in rodent models [30] and theknown pathogenesis of dry eye in which tear film hyperosmolalityproduces downstream pathology [31,32] support the utility of CFTRactivator therapy in human dry eye.

In conclusion, CFTR activator therapy is predicted to have broadclinical indications for prosecretory therapy of dry eye caused by avariety of etiologies, including ‘idiopathic’ dry eye. Because of theirunique mechanism of action CFTR activators are combinable with approvedimmunosuppressants and possible future therapeutics such as ENaCinhibitors. Compared with immunosuppressant drugs, CFTR activatortherapy rescues an early, initiating event in dry eye pathogenesis in anetiology-agnostic manner. The aminophenyl-1,3,5-triazines synthesizedand tested herein are candidate lead compounds for further preclinicaldevelopment.

Experimental Details and Data

Synthesis Procedures

General Procedures. All solvents and chemicals were used as purchasedwithout further purification. Reaction progress was monitored on Merckpre-coated silica gel plates (with fluorescence at 254 nm) using ethylacetate/n-hexane as solvent system. Column chromatography was done withFluka silica gel 60 (230-400 mesh). Proton (¹H) and carbon (¹³C) NMRspectra were recorded on a Bruker Avance 300 or 500 (500.13 MHz for ¹H;125.76 MHz for ¹³C) using CDCl₃ (δ 7.26), CD₃OD (δ=4.87 and 3.31),acetone-d₆ (δ 2.05), or DMSO-d₆ (δ 2.5) as solvents. Chemical shifts aregiven in parts per million (ppm) (6 relative to residual solvent peakfor ¹H and ¹³C). HRMS was performed using a hybrid quadrupole Orbitrapmass analyzer, QExactive (Thermo, Bremen, Germany), with an electrosprayionization source. The mass resolution was set as 70,000 at m/z 200 andthe mass accuracy was less than 3 ppm. Purity of all final compounds was95% or higher. Below we report synthesis and analytic data forintermediates and representative final compounds 1 and 12. Analytic datafor 6a-6k, 7a-7g, and 11 are reported in Supporting Information.

2,4-Dichloro-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazine(2). To a solution of cyanuric chloride (2 g, 10.84 mmol) in THF (50 ml)at −80° C. was added potassium carbonate (3.0 g, 21.74 mmol). A solutionof 1,1,1,3,3,3-hexafluoro-2-propanol (1.36 ml, 10.30 mmol) in THF (20ml) was then added dropwise over 30 min. The mixture was stirred foradditional 30 min at −80° C. and quenched with water. The reactionmixture was diluted with EtOAc and washed with 1N HCl solution. Theorganic layer was washed with brine and dried over MgSO₄. The solventwas removed under reduced pressure to afford a white solid (1.5 g, 62%).The residue was used for further reaction without purification.

2,4-Dichloro-6-[(2,2,3,3-tetrafluoropropan)oxy]-1,3,5-triazine (3). To asolution of cyanuric chloride (2 g, 10.84 mmol) in THF (50 ml) at −80°C. was added potassium carbonate (3 g, 21.74 mmol). A solution of2,2,3,3-tetrafluoropropanol (1.2 ml, 10.3 mmol) in THF (20 ml) was thenadded dropwise over 30 min. The mixture was stirred for additional 30min at −80° C. and quenched with water. The reaction mixture was dilutedwith EtOAc and the organic layer was washed with brine and dried overMgSO₄. The solvent was removed under reduced pressure to afford a whitesolid (1.3 g, 68%). The residue was used for further reaction withoutpurification. ¹H NMR (300 MHz, CDCl₃): δ=6.01 (tt, 1H, J=3.96, 52.9 Hz),4.90-4.79 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ=168.5, 165.1, 165.0,138.4, 128.7, 123.4, 122.7, 120.3, 119.0, 68.3 (t, 34 Hz), 42.4, 42.0,12.9, 12.8; LRMS-ESI: m/z 279.

4-Chloro-N,N-diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-1,3,5-triazin-2-amine(4). To a solution of 2 (1.6 g, 5.06 mmol) in THF (40 ml) was addeddiisopropylethylamine (1.06 ml, 6.08 mmol) and diethylamine (0.5 ml, 5.0mmol) at 0° C. The mixture was stirred for 1 h at 0° C., then quenchedwith water. The reaction mixture was extracted with EtOAc, washed withwater and brine, and dried over MgSO₄. The solvent was removed underreduced pressure to afford a white solid (950 mg, 55%).

4-Chloro-N,N-diethyl-6-[(2,2,3,3,-tetrafluoropropan)oxy]-1,3,5-triazin-2-amine(5). To a solution of 3 (1.3 g, 5 mmol) in THF (30 ml) was addeddiisopropylethylamine (1 ml, 6 mmol) and diethylamine (0.5 ml, 5.0 mmol)at 0° C. The mixture was stirred for 1 h at 0° C., then quenched withwater. The reaction mixture was extracted with EtOAc, washed with waterand brine, and dried over MgSO4. The solvent was removed under reducedpressure to afford yellow oil (900 mg, 57%).

Analogs 6a-6k and 7a-7g were prepared as described for 1, with varioussubstituted anilines and 4 or 5.

N,N-Diethyl-6-[(1,1,1,3,3,3-hexafluoropropan-2-yl)oxy]-N-phenyl-1,3,5-triazine-2,4-diamine(1). To a solution of 4 (500 mg, 1.42 mmol) in THF (30 ml) was addeddiisopropylethylamine (0.49 ml, 2.83 mmol) and aniline (0.15 ml, 1.7mmol). The mixture was refluxed for 16 h. After cooling to roomtemperature the reaction mixture was extracted with EtOAc, washed withbrine and dried over MgSO₄. The solvent was removed under reducedpressure and purified by flash chromatography (1:10 EtOAc/Hex) to afford1 as a white powder (280 mg, 48%). ¹H NMR (300 MHz, CDCl₃): δ=7.61 (d,2H, J=7.8 Hz), 7.55 (brs, 1H), 7.35 (t, 1H, J=7.5 Hz), 7.11 (t, 1H,J=7.4 Hz), 6.37 (sep, 1H, J=6.2 Hz), 3.69-3.58 (m, 4H), 1.29-1.20 (m,6H); ¹³C NMR (75 MHz, CDCl₃): δ=168.5, 165.1, 165.0, 138.4, 128.7,123.4, 122.7, 120.3, 119.0, 68.3 (t, 34 Hz), 42.4, 42.0, 12.9, 12.8;HRMS (ESI): m/z calculated for C₁₆H₁₈F₆N₅O [M+H⁺]: 410.1410. Found:410.1411.

4,6-Dichloro-N-methyl-1,3,5-triazine-2-amine (8)

To a solution of cyanuric chloride (5 g, 27.3 mmol) in acetone (30 ml)at 0° C. was added methylamine (13.7 ml, 2 M in THF) dropwise over 30min. The mixture was stirred for 1 h at 0° C. and then crushed ice wasadded. The resulting precipitate was filtered and washed with water toafford 8 as a white solid (3.7 g, 75.7%), which was used for furtherreaction without purification. ¹H NMR (300 MHz, CDCl₃) δ=6.21-5.81 (m,1H), 4.81-4.67 (m, 2H), 3.06-3.03 (m, 3H); ¹³C NMR (75 MHz, CDCl₃)δ=170.8, 169.2, 168.4, 108.9, 63.3, 25.5.

4-Chloro-6-((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)-N-methyl-1,3,5-triazin-2-amine(9). To a solution of 8 (2 g, 11.17 mmol) in THF (40 ml) at 0° C. wasadded potassium carbonate (11.56 g, 83.8 mmol) and1,1,1,3,3,3-hexafluoro-2-propanol (1.4 ml, 13.4 mmol). The mixture wasstirred overnight at ambient temperature. The reaction mixture wasdiluted with EtOAc, and then organic layer was washed with brine anddried over MgSO₄. The solvent was removed under reduced pressure. Awhite solid was obtained (1.4 g, 43%).

4-Chloro-6-[(2,2,3,3-tetrafluoropropan)oxy]N-methyl-1,3,5-triazine-2-amine(10). To a solution of 8 (630 mg, 3.52 mmol) in THF (30 ml) at 0° C. wasadded potassium carbonate (970 mg, 7.04 mmol). A solution of2,2,3,3-tetrafluoropropanol (0.31 ml, 3.52 mmol) in THF (20 ml) wasadded dropwise over 30 min. The mixture was stirred for additional 30min at 0° C., then quenched with water. The reaction mixture was dilutedwith EtOAc, and the organic layer was washed with brine and dried overMgSO₄. Solvent was removed under reduced pressure to afford a whitesolid (640 mg, 66%). The residue was used for further reaction withoutpurification. ¹H NMR (300 MHz, CDCl₃): δ=6.98-5.79 (m, 1H), 4.87-4.69(m, 2H), 3.05 (d, 3H).

N-methyl-N′-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine(12). To a solution of 10 (70 mg, 0.26 mmol) in THF (3 ml) was addeddiisopropylethylamine (0.09 ml, 0.51 mmol) and aniline (0.03 ml, 0.31mmol). The mixture was refluxed for 16 h. After cooling to roomtemperature the reaction mixture was extracted with EtOAc, washed withbrine and dried over MgSO₄. Solvent was removed under reduced pressureand purified by flash chromatography (1:2 EtOAc/Hex) to afford 12 as awhite powder (mg, 74%). ¹H NMR (300 MHz, CDCl₃): δ=7.61 (d, 2H, J=7.8Hz), 7.55 (brs, 1H), 7.35 (t, 1H, J=7.5 Hz), 7.11 (t, 1H, J=7.4 Hz),6.37 (sep, 1H, J=6.2 Hz), 3.69-3.58 (m, 4H), 1.29-1.20 (m, 6H); ¹³C NMR(75 MHz, CDCl₃): δ=168.5, 165.1, 165.0, 138.4, 128.7, 123.4, 122.7,120.3, 119.0, 68.3 (t, 34 Hz), 42.4, 42.0, 12.9, 12.8; HRMS (ESI): m/zcalculated for C₁₃H4F₄N₅O [M+H+]: 332.1129. Found: 332.1127.

Mice. Wildtype and CF (homozygous ΔF508-CFTR mutant) mice in a CD1genetic background were bred at the University of California SanFrancisco (UCSF) Animal Facility. Mice aged 8 to 12 weeks (25 to 35 g)were used. Animal protocols were approved by the UCSF InstitutionalAnimal Care and Use Committee and were in compliance with the ARVOStatement for the Use of Animals in Ophthalmic and Visual Research.

Cell Culture. Fischer Rat Thyroid (FRT) cells stably co-expressing humanwildtype CFTR or TMEM16A and the halide-sensitive yellow fluorescentprotein (YFP)-H148Q were cultured as described [15,33]. HT-29 expressingYFP were cultured as described [34]. Primary cultures of human bronchialepithelial (HBE) cells were maintained at an air-liquid interface asdescribed previously [35].

Platereader assays of chloride channel function. CFTR activity wasassayed as described [15]. FRT cells co-expressing YFP and wildtype CFTRwere washed with phosphate-buffered saline (PBS) and then incubated for10 min with test compounds in PBS containing 125 nM forskolin. I⁻ influxwas measured in a plate reader with initial baseline read for 2 s andthen for 12 s after rapid addition of an I⁻-containing solution. TMEM16Aactivity was assayed similarly as described [33] using FRT cellsco-expressing YFP and TMEM16A. Activity of non-TMEM16A CaCC was assayedas described [34] in HT-29 cells expressing YFP. In each assay initialrates of I⁻ influx were computed as a linear measure of channelfunction.

Short-circuit current measurement. Short-circuit current measurementswere done as described [9]. Briefly, Snapwell (Corning Costar, Corning,N.Y.) inserts containing CFTR_(act)-expressing FRT cells or humanbronchial epithelial cells were mounted in Ussing chambers(Physiological Instruments, San Diego, Calif.). For FRT cells thehemichambers were filled with 5 m1 of HCO₃ ⁻-buffered solution(basolateral) and half-Cl⁻ solution (apical), and the basolateralmembrane was permeabilized with 250 μg/ml amphotericin B. SymmetricalHCO₃ ⁻-buffered solutions were used for human bronchial epithelialcells. Solutions were bubbled with 95% O₂/5% CO₂ and maintained at 37°C., and short-circuit current was measured on a DVC-1000 voltage clamp(World Precision Instruments Inc., Sarasota, Fla.) using Ag/AgClelectrodes and 3 M KCl agar bridges.

cAMP and Ca²⁺ assays. FRT cells expressing wildtype CFTR were culturedin 12-well plates. After washing with PBS, cells were incubated for 10min with compounds in the absence or presence of 90 nM forskolin andthen lysed. Lysates were assayed for cAMP content using a cAMPimmunoassay kit (Parameter cAMP Immunoassay Kit, R&D Systems,Minneapolis, Minn.). For cytoplasmic calcium measurements, FRT cells in96-well black-walled microplates were loaded with Fluo-4 NW (Invitrogen,Carlsbad, Calif.) and Fluo-4 fluorescence was measured with a platereader equipped with syringe pumps and monochromators.

Cytotoxicity. FRT cells were cultured overnight in black 96-well Costarmicroplates and incubated with test compounds at up to 10 μM (themaximum solubility in PBS) for 8 h. Cytotoxicity was measured by AlamarBlue assay (Invitrogen, Carlsbad, Calif.).

Tear volume. Tear volume was measured using phenol red threads(Zone-Quick, Oasis Medical, Glendora, Calif.) as described [36]. Threadswere placed for 10 s in the lateral canthi of isofluorane-anesthetizedmice using jewelers' forceps. Tear volume was determined from the lengthof thread wetting as visualized under a dissecting microscope. Serialmeasurements were done to evaluate compound pharmacodynamics aftertopical application of a single, 2.5-μL drop of ophthalmic formulationcontaining 0-100 μM of 1 or 12 in Ringer's solution with 0.3%carboxymethylcellulose (high viscosity, VWR, Radnor, Pa.), 0.015%benzalkonium chloride preservative, and 1% DMSO. Both eyes in each mousereceived the same treatment to control for potential contralateraleffects of a given treatment.

In vitro metabolic stability. Compounds (each 5 μM) were incubated forspecified times at 37° C. with rat liver microsomes (1 mg/ml;Sigma-Aldrich) in potassium phosphate buffer containing 1 mM NADPH, asdescribed¹⁴. Ice-cold EtOAc was then added and the mixture wascentrifuged for 15 min at 3000 rpm. The supernatant was analyzed bylow-resolution mass spectrometry using a LC/MS system (Waters 2695 HPLCwith Micromass ZQ). LC was done on a Xterra MS C18 column (2.1 mm×100mm, 3.5 μm) with 0.2 mL/min water/acetonitrile (containing 0.1% formicacid), 12 min linear gradient, 5 to 95% acetonitrile. UV absorbance wasdetected at 254 nm.

Statistics. Statistical analysis was performed using Prism 5 GraphPadSoftware package (San Diego, Calif.). Serial tear volume measurementswere analyzed by two-way ANOVA with Dunnett post-hoc analysis. Data arepresented as mean±standard error of the mean (S.E.M.), and statisticalsignificance was set at p<0.01 (*).

Abbreviations. CFTR, cystic fibrosis transmembrane conductanceregulator; cAMP, cyclic adenosine monophosphate; YFP, yellow fluorescentprotein; CF, cystic fibrosis; FRT cells, Fischer rat thyroid cells;LC/MS, liquid chromatography/mass spectrometry; fsk, forskolin; SAR,structure-activity relationships.

References (Example 6)

-   [1] Verkman, A. S.; Galietta, L. J. Chloride channels as drug    targets. Nat. Rev. Drug Disc. 2009, 8, 153-171; [2] Schmidt, B. Z.;    Haaf, J. B.; Leal, T.; Noel, S. Cystic fibrosis transmembrane    conductance regulator modulators in cystic fibrosis: current    perspectives. Clin. Pharmacol. 2016, 21, 127-140; [3]    Thiajarajah, J. R.; Donowitz, M.; Verkman, A. S. Secretory diarrhea:    mechanisms and emerging therapies. Nature Rev. Gastroenterol.    Hepatol. 2015, 12, 446-457; [4] Ong, T.; Ramsey, B. W. New    rherapeutic approaches to modulate and correct cystic fibrosis    transmembrane conductance regulator. Pediatr. Clin. North. Am. 2016,    63, 751-764; [5] Chao, A. C.; de Sauvage, F. J.; Dong, Y. J.;    Wagner, J. A; Goeddel, D. V.; Gardner, P. Activation of intestinal    CFTR Cl— channel by heat-stable enterotoxin and guanylin via    cAMP-dependent protein kinase. EMBO J. 1994, 13, 1065-1072; [6]    Moon, C.; Zhang, W.; Sundaram, N.; Yarlagadda, S.; Reddy, V. S.;    Arora, K.; Helmrath, M. A.; Naren, A. P. Drug-induced secretory    diarrhea: A role for CFTR. Pharmacol. Res. 2015, 102, 107-112; [7]    Field, M.; Mechanisms of action of cholera and Escherichia coli    enterotoxins. Am. J. Clin. Nutr. 1979, 32, 189-196; [8] Solomon G.    M.; Marshall, S. G.; Ramsey, B. W.; Rowe, S. M. Breakthrough    therapies: Cystic fibrosis (CF) potentiators and correctors.    Pediatr. Pulmonol. 2015, 50, S3-S13; [9] Cil, O.; Phuan, P. W.; Lee,    S.; Tan, J.; Haggie, P. M.; Levin, M. H.; Sun, L.; Thiagarajah, J.    R.; Ma, T.; Verkman, A. S. CFTR activator increases intestinal fluid    secretion and normalizes stool output in a mouse model of    constipation. Cell. Mol. Gastroentrol. Hepatol. 2016, 2, 317-327;    [10] Cil, O.; Phuan, P. W.; Son, J. H.; Zhu, J. S.; Ku, C. K.;    Tabib, N. A.; Teuthorn, A. P.; Ferrera, L.; Zachos, N. C.; Lin, R.;    Galietta, L. J.; Donowitz, M.; Kurth, M. J.; Verkman, A. S.    Phenylquinoxalinone CFTR activator as potential prosecretory therapy    for constipation. Transl. Res. 2016, (in press); [11] Solomon, G.    M.; Raju, S. V.; Dransfield, M. T.; Rowe, S. M. Therapeutic    approaches to acquired cystic fibrosis transmembrane conductance    regulator dysfunction in chronic bronchitis. Ann. Am. Thorac. Soc.    2016, 13, Suppl 2:S169-S176; [12] Flores, A. M.; Casey, S. D.;    Felix, C. M.; Phuan, P. W.; Verkman, A. S.; Levin, M. H.    Small-molecule CFTR activators increase tear secretion and prevent    experimental dry eye disease. FASEB J. 2016, 30, 1789-1797; [13]    Cil, O.; Phuan, P. W.; Gillespie, A. M.; Lee, S.; Tradtrantip, L.;    Yin, J.; Tse, M.; Zachos, N. C.; Lin, R; Donowitz, M.;    Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor    (R)-BPO-27 for antisecretory therapy of diarrheas caused by    bacterial enterotoxins. FASEB J. 2016 (in press); [14] Snyder, D.    S.; Tradtrantip, L.; Yao, C.; Kurth, M. J.; Verkman, A. S. Potent,    metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR    inhibitors for polycystic kidney disease. J. Med. Chem. 2011, 54,    5468-5477; [15] Ma, T.; Vetrivel, L.; Yang, H.; Pedemonte, N.;    Zegarra-Moran, O.; Galietta, L. J.; Verkman, A. S. High-affinity    activators of CFTR chloride conductance identified by    high-throughput screening. J. Biol. Chem. 2002, 277, 37235-37241;    [16] Schaumberg, D. A.; Dana, R., Buring, J. E.; Sullivan, D. A.    Prevalence of dry eye disease among US men: estimates from the    Physicians' Health Studies. Arch. Ophthalmol. 2009, 127, 763-768;    [17] Schaumberg, D. A.; Sullivan, D. A.; Buring, J. E.; Dana, M. R.    Prevalence of dry eye syndrome among US women. Am. J. Ophthalmol.    2003, 136, 318-326; [18] Alves, M.; Foseca, E. C.; Alves, M. F.;    Malki, L. T.; Arruda, G. V.; Reinach, P. S.; Rocha, E. M. Dry eye    disease treatment: a systematic review of published trials and    critical appraisal of therapeutic strategies. Ocul. Surf 2013, 11,    181-192; [19] Sheppard, J. D.; Torkildsen, G. L.; Lonsdale, J. D.;    D'Ambrosio Jr. F. A.; McLaurin, E. B.; Eiferman, R. A.; Kennedy, K.    S.; Semba, C. P.; OPUS-1 Study Group. Lifitegrast ophthalmic    solution 5.0% for treatment of dry eye disease: results of the    OPUS-1 phase 3 study. Ophthalmology 2014, 121, 475-483; [20] Jowa,    L.; Howd, R. Should atrazine and related chlorotriazines be    considered carcinogenic for human health risk assessment? J.    Environ. Sci. Health C Environ. Carcinog. Ecotoxicol Rev. 2011, 29,    91-144; [21] Moon, H.-S.; Jacobson, E. M.; Khersonsky, S. M.;    Luzung, M. R.; Walsh, D. P.; Xiong, W.; Lee J. W.; Parikh, P. B.;    Lam, J. C.; Kang, T.-W.; Rosania, G. R; Schier, A. F.; Chang, Y.-T.    A novel microtubule destabilizing entity from orthogonal synthesis    of triazine library and zebrafish embryo screening. J. Am. Chem.    Soc. 2002, 124, 11608-11609; [22] Plebanek, E.; Chevrier, F.; Roy,    V.; Garnne, T.; Lecaille, F.; Warszycki, D.; Bojarski, A. J.;    Lalmanach, G.; Agrofoglio, L. A. Straightforward synthesis of    2,4,6-trisubstituted 1,3,5-triazine compounds targeting cysteine    cathepsins K and S. Eur. J. Med. Chem. 2016, 121, 12-20; [23]    Kompella, U. B.; Kim, K. J.; Lee, V. H. Active chloride transport in    the pigmented rabbit conjunctiva. Curr. Eye Res. 1993, 12,    1041-1048; [24] Turner, H. C.; Bemstein, A.; Candia, O. A. Presence    of CFTR in the conjunctival epithelium. Curr. Eye Res. 2002, 24,    182-187; [25] Al-Nakkash, L.; Reinach, P. S. Activation of a    CFTR-mediated chloride current in a rabbit corneal epithelial cell    line. Invest. Ophthalmol. Vis. Sci. 2001, 42, 2353-2370; [26]    Shiue, M. H.; Gukasyan, H. J; Kim, K. J.; Loo, D. D.; Lee, V. H.    Characterization of cyclic AMP-regulated chloride conductance in the    pigmented rabbit conjunctival epithelial cells. Can. J. Physiol.    Pharmacol. 2002, 80, 533-540; Levin, M. H.; Verkman, A. S.    CFTR-regulated chloride transport at the ocular surface in living    mice measured by potential differences. Invest. Ophthalmol. Vis.    Sci. 2005, 46, 1428-1434; [28] Levin, M. H.; Kim, J. K.; Hu, J.;    Verkman A. S. Potential difference measurements of ocular surface    Na⁺ absorption analyzed using an electrokinetic model. Invest.    Ophthalmol. Vis. Sci. 2006, 47, 306-316; [29] Yu, D.; Thelin, W. R.;    Rogers, T. D.; Stutts, M. J.; Randell, S. H.; Grubb, B. R.;    Boucher, R. C. Regional differences in rat conjunctival ion    transport activities. Am. J. Physiol. Cell Physiol. 2012, 303,    C₇₆₇-780; [30] Thelin, W. R.; Johnson, M. R.; Hirsh, A. J.;    Kublin, C. L.; Zoukhri, D. J. Effect of topically applied epithelial    sodium channel inhibitors on tear production in normal mice and in    mice with induced aqueous tear deficiency. Ocul. Pharmacol. Ther.    2012, 28, 433-438; [31] Gilbard, J. P.; Carter, J.; Sang, D.;    Refojo, M.; Hanninen, L.; Kenyon, K. R. Morphologic effect of    hyperosmolarity on rabbit corneal epithelium. Ophthalmology 1984,    91, 1205-1212; [32] Liu, H.; Begley, C.; Chen, M.; Bradley, A.;    Bonanno, J.; McNamara, N.; Nelson, J.; Simpson, T. A link between    tear instability and hyperosmolarity in dry eye. Invest. Ophthalmol.    Vis. Sci. 2009, 50, 3671-3679; [33] Namkung, W.; Phuan, P. W.;    Verkman, A. S. TMEM16A inhibitors reveal TMEM16A as a minor    component of calcium-activated chloride channel conductance in    airway and intestinal epithelial cells. J. Biol. Chem. 2011, 286,    2365-2374; [34] De La Fuente, R.; Namkung, W.; Mills, A.;    Verkman, A. S. Small-molecule screen identifies inhibitors of a    human intestinal calcium-activated chloride channel. Mol. Pharmacol.    2008, 73, 758-768; [35] Haggie, P. M.; Phuan, P. W.; Tan, J. A.;    Zlock, L.; Finkbeiner, W. E.; Verkman, A. S. Inhibitors of pendrin    anion exchange identified in a small molecule screen increase airway    surface liquid volume in cystic fibrosis. FASEB J. 2016, 30,    2187-2197; [36] Stewart, P.; Chen, Z; Farley, W.; Olmos, L.;    Pflugfelder, S. C. Effect of experimental dry eye on tear sodium    concentration in the mouse. Eye Contact Lens 2005, 31, 175-178;

What is claimed is:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—; R² isC₂-C₄ haloalkyl substituted with at least four fluorines, substitutedheteroalkyl, or unsubstituted heteroalkyl; R³ is hydrogen; R⁴ issubstituted or unsubstituted alkyl; R⁵ is hydrogen; and R¹, R⁶, R⁷, R⁸and R⁹ are each hydrogen.
 2. The compound of claim 1, wherein R⁴ is —CH₃or —CH₂CH₃.
 3. The compound of claim 1, wherein R² is C₂-C₄ haloalkylsubstituted with at least four fluorines.
 4. The compound of claim 3,wherein R² is —CH(CF₃)₂ or —CH₂—CF₂—CHF₂.
 5. The compound of claim 1,wherein the compound of Formula I is:


6. A compound that is:

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim6, wherein the compound is:


8. The compound of claim 6, wherein the compound is a pharmaceuticallyacceptable salt of:


9. A pharmaceutical composition, comprising a pharmaceuticallyacceptable excipient, and a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is —O—; R² isC₂-C₄ haloalkyl substituted with at least four fluorines, substitutedheteroalkyl; or unsubstituted heteroalkyl; R³ is hydrogen; R⁴ issubstituted or unsubstituted alkyl; R⁵ is hydrogen; and R¹, R⁶, R⁷, R⁸and R⁹ are hydrogen.
 10. The pharmaceutical composition of claim 9,wherein R⁴ is —CH₃ or —CH₂CH₃.
 11. The pharmaceutical composition ofclaim 9, wherein R² is C₂-C₄ haloalkyl substituted with at least fourfluorines.
 12. The pharmaceutical composition of claim 11, wherein R² is—CH(CF₃)₂ or —CH₂—CF₂—CHF₂.
 13. The pharmaceutical composition of claim9, wherein the compound of Formula I is:


14. A pharmaceutical composition comprising a compound that is:

or a pharmaceutically acceptable salt thereof.
 15. The pharmaceuticalcomposition of claim 14, wherein the compound is:


16. The pharmaceutical composition of claim 14, wherein the compound isa pharmaceutically acceptable salt of: